Anthracycline disulfide intermediates, antibody-drug conjugates and methods

ABSTRACT

The invention provides antibody-drug conjugates comprising an antibody conjugated to an anthracycline drug moiety via a disulfide linker, anthracycline disulfide intermediates, and methods of using the antibody-drug conjugates.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 14/851,206, filed 11Sep. 2015, which claims the benefit under 35 USC § 119(e) of U.S.Provisional Application Ser. No. 62/049,720 filed on 12 Sep. 2014. Theentire content of the applications referenced above are herebyincorporated by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Sep. 4, 2015, isnamed P05813-US_SL.txt and is 16,012 bytes in size.

FIELD OF THE INVENTION

The invention relates generally to antibodies conjugated toanthracycline disulfide intermediates to form antibody-drug conjugateswith therapeutic or diagnostic applications. The antibodies may beengineered with free cysteine amino acids, reactive for conjugation withthe anthracycline disulfide intermediates. The invention also relates tomethods of using the antibody-drug conjugate compounds for in vitro, insitu, and in vivo diagnosis or treatment of mammalian cells, orassociated pathological conditions.

BACKGROUND OF THE INVENTION

Antibody drug conjugates (ADC) are targeted chemotherapeutic moleculescombining the properties of both antibodies and cytotoxic drugs bytargeting potent cytotoxic drugs to antigen-expressing tumor cells,internalization, and release of drug, thereby enhancing their anti-tumoractivity (Carter, P. and Senter, P. (2008) The Cancer Jour.14(3):154-169). Successful ADC development for a given target antigendepends on optimization of antibody selection, linker design andstability, cytotoxic drug potency and mode of drug and linkerconjugation to the antibody (Polakis, P. (2005) Current Opinion inPharmacology 5:382-387).

KADCYLA® (trastuzumab emtansine, Genentech) has recently been approvedfor the treatment of breast cancer. Trastuzumab emtansine is anantibody-drug conjugate with a maytansine drug moiety DM1 covalentlyattached via a stable, non-disulfide linker MCC to the anti-HER2antibody trastuzumab (Phillips G. et al. (2008) Cancer Res. 68:9280-90).Trastuzumab is the antibody formulated as HERCEPTIN® (Genentech),approved in 1998 for HER2 positive breast cancer therapy.

Anthracyclines are used in the treatment of numerous cancers such asleukemia, breast carcinoma, lung carcinoma, ovarian adenocarcinoma andsarcomas. Commonly used anthracyclines include doxorubicin, epirubicin,idarubicin and daunomycin. Morpholino analogs of doxorubicin anddaunorubicin, formed by cyclization on the glycoside amino group, havegreater potency (Acton et al (1984) J. Med. Chem. 638-645; U.S. Pat. No.4,464,529; U.S. Pat. No. 4,672,057; U.S. Pat. No. 5,304,687).Nemorubicin is a semisynthetic analog of doxorubicin with a2-methoxymorpholino group on the glycoside amino of doxorubicin and hasbeen under clinical evaluation (Grandi et al (1990) Cancer Treat. Rew.17:133; Ripamonti et al (1992) Brit. J. Cancer 65:703), including phaseII/III trials for hepatocellular carcinoma (Sun et al (2003) Proceedingsof the American Society for Clinical Oncology 22, Abs1448; Quintieri(2003) Proceedings of the American Association of Cancer Research,44:1st Ed, Abs 4649; Pacciarini et al (2006) Jour. Clin. Oncology24:14116). Immunoconjugates and prodrugs of daunorubicin and doxorubicinhave been prepared and studied (Kratz et al (2006) Current Med. Chem.13:477-523; Jeffrey et al (2006) Bioorganic & Med. Chem. Letters16:358-362; Torgov et al (2005) Bioconj. Chem. 16:717-721; Nagy et al(2000) Proc. Natl. Acad. Sci. 97:829-834; Dubowchik et al (2002) Bioorg.& Med. Chem. Letters 12:1529-1532; King et al (2002) J. Med. Chem.45:4336-4343; U.S. Pat. No. 6,630,579). The antibody-drug conjugateBR96-doxorubicin reacts specifically with the tumor-associated antigenLewis-Y and has been evaluated in phase I and II studies (Saleh et al(2000) J. Clin. Oncology 18:2282-2292; Ajani et al (2000) Cancer Jour.6:78-81; Tolcher et al (1999) J. Clin. Oncology 17:478-484).

Several metabolites of nemorubicin (MMDX) from liver microsomes havebeen characterized, including PNU-159682, (Quintieri et al (2005)Clinical Cancer Research, 11(4):1608-1617; Beulz-Riche et al (2001)Fundamental & Clinical Pharmacology, 15(6):373-378; EP 0889898; WO2004/082689; WO 2004/082579). PNU-159682 was remarkably more cytotoxicthan nemorubicin and doxorubicin in vitro, and was effective in vivotumor models. PNU-159682 is named as3′-deamino-3″,4′-anhydro-[2″(S)-methoxy-3″(R)-oxy-4″-morpholinyl]doxorubicin,and has the structure:

Certain PNU-159682 antibody-drug conjugates have been described (WO2009/099741; WO 2010/009124; U.S. Pat. No. 8,742,076; U.S. Pat. No.8,389,697). Processes for making PNU-159682 analogs are described (U.S.Pat. No. 8,470,984).

SUMMARY

The invention includes anthracycline disulfide intermediates havingFormula I:

Ant-L-(Z)_(m)—X  I

wherein Ant is selected from the structure:

where the wavy line indicates the attachment to L;

L is a linker selected from —CH₂O—, —CH₂N(R)—, —N(R)—, —N(R)(C₁-C₁₂alkylene)-, —N(R)(C₂-C₈ alkenylene)-, —N(R)(C₂-C₈ alkynylene)-,—N(R)(CH₂CH₂O)_(n)—, and the structure:

where the wavy lines indicate the attachments to Ant and Z; and

Z is an optional spacer selected from —CH₂C(O)—, —CH₂C(O)NR(C₁-C₁₂alkylene)-, and the structure:

where the wavy lines indicate the attachments to L and X;

R is H, C₁-C₁₂ alkyl, or C₆-C₂₀ aryl;

Z¹ is selected from —(C₁-C₁₂ alkylene), —(C₂-C₈ alkenylene)-, —(C₂-C₈alkynylene)-, —O(C₁-C₁₂ alkylene)-, —O(C₂-C₈ alkenylene)-, —O(C₂-C₈alkynylene)-, and —(CH₂CH₂O)_(n)—;

m is 0 or 1;

n is 1 to 6;

X is pyridyl disulfide where pyridyl is optionally substituted with oneor more groups selected from NO₂, Cl, F, and Br; and

alkylene, alkenylene, alkynylene, alkyl, and aryl are optionallysubstituted with one or more groups selected from F, Cl, Br, N(CH₃)₂,and OCH₃.

The invention includes anthracycline disulfide intermediates covalentlyattached to antibodies to form antibody-drug conjugate (ADC) compoundswith therapeutic or diagnostic applications.

Another aspect of the invention is an antibody-drug conjugate compoundcomprising an antibody covalently attached by a disulfide, linker L andan optional spacer Z to one or more anthracycline derivative drugmoieties D, the compound having Formula II:

Ab-S—S—(Z_(m)-L-D)_(p)  II

or a pharmaceutically acceptable salt thereof, wherein:

Ab is an antibody which binds to one or more tumor-associated antigensor cell-surface receptors selected from (1)-(53):

(1) BMPR1B (bone morphogenetic protein receptor-type IB);

(2) E16 (LAT1, SLC7A5);

(3) STEAP1 (six transmembrane epithelial antigen of prostate);

(4) MUC16 (0772P, CA125);

(5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin);

(6) Napi2b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodiumphosphate), member 2, type II sodium-dependent phosphate transporter3b);

(7) Sema 5b (FLJ0372, KIAA1445, Mm.42015, SEMA5B, SEMAG, Semaphorin 5bHlog, sema domain, seven thrombospondin repeats (type 1 and type1-like), transmembrane domain (TM) and short cytoplasmic domain,(semaphorin) 5B);

(8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12, RIKENcDNA 2700050C12 gene);

(9) ETBR (Endothelin type B receptor);

(10) MSG783 (RNF124, hypothetical protein F1120315);

(11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostatecancer associated gene 1, prostate cancer associated protein 1, sixtransmembrane epithelial antigen of prostate 2, six transmembraneprostate protein);

(12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptorpotential cation channel, subfamily M, member 4);

(13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derivedgrowth factor);

(14) CD21 (CR2 (Complement receptor 2) or C3DR (C3d/Epstein Barr virusreceptor) or Hs 73792);

(15) CD79b (CD79B, CD7913, IGb (immunoglobulin-associated beta), B29);

(16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing phosphataseanchor protein 1a), SPAP1B, SPAP1C);

(17) HER2;

(18) NCA;

(19) MDP;

(20) IL20Rα;

(21) Brevican;

(22) EphB2R;

(23) ASLG659;

(24) PSCA;

(25) GEDA;

(26) BAFF-R (B cell-activating factor receptor, BLyS receptor 3, BR3);

(27) CD22 (B-cell receptor CD22-B isoform);

(28) CD79a (CD79A, CD79α, immunoglobulin-associated alpha);

(29) CXCR5 (Burkitt's lymphoma receptor 1);

(30) HLA-DOB (Beta subunit of MEW class II molecule (Ia antigen));

(31) P2X5 (Purinergic receptor P2X ligand-gated ion channel 5);

(32) CD72 (B-cell differentiation antigen CD72, Lyb-2);

(33) LY64 (Lymphocyte antigen 64 (RP105), type I membrane protein of theleucine rich repeat (LRR) family);

(34) FcRH1 (Fc receptor-like protein 1);

(35) FcRH5 (IRTA2, Immunoglobulin superfamily receptor translocationassociated 2);

(36) TENB2 (putative transmembrane proteoglycan);

(37) PMEL17 (silver homolog; SILV; D12S53E; PMEL17; SI; SIL);

(38) TMEFF1 (transmembrane protein with EGF-like and twofollistatin-like domains 1; Tomoregulin-1);

(39) GDNF-Ra1 (GDNF family receptor alpha1; GFRA1; GDNFR; GDNFRA; RETL1;TRNR1; RET1L; GDNFR-alpha1; GFR-ALPHA-1);

(40) Ly6E (lymphocyte antigen 6 complex, locus E; Ly67, RIG-E, SCA-2,TSA-1);

(41) TMEM46 (shisa homolog 2 (Xenopus laevis); SHISA2);

(42) Ly6G6D (lymphocyte antigen 6 complex, locus G61); Ly6-D, MEGT1);

(43) LGR5 (leucine-rich repeat-containing G protein-coupled receptor 5;GPR49, GPR67);

(44) RET (ret proto-oncogene; MEN2A; HSCR1; MEN2B; MTC1; PTC; CDHF12;Hs.168114; RET51; RET-ELE1);

(45) LY6K (lymphocyte antigen 6 complex, locus K; LY6K; HSJ001348;FLJ35226);

(46) GPR19 (G protein-coupled receptor 19; Mm.4787);

(47) GPR54 (KISS1 receptor; KISS1R; GPR54; HOT7T175; AXOR12);

(48) ASPHD1 (aspartate beta-hydroxylase domain containing 1; LOC253982);

(49) Tyrosinase (TYR; OCAIA; OCA1A; tyrosinase; SHEP3);

(50) TMEM118 (ring finger protein, transmembrane 2; RNFT2; FLJ14627);

(51) GPR172A (G protein-coupled receptor 172A; GPCR41; FLJ11856;D15Ertd747e);

(52) CD33; and

(53) CLL-1;

D is an anthracycline derivative selected from the structure:

where the wavy line indicates the attachment to L;

L is a linker selected from —CH₂O—, —CH₂N(R)—, —N(R)—, —N(R)(C₁-C₁₂alkylene)-, —N(R)(C₂-C₈ alkenylene)-, —N(R)(C₂-C₈ alkynylene)-,—N(R)(CH₂CH₂O)_(n)—, and the structure:

where the wavy lines indicate the attachments to D and Z; and

Z is an optional spacer selected from —CH₂C(O)—, —CH₂C(O)NR(C₁-C₁₂alkylene)-, and the structures:

R is H, C₁-C₁₂ alkyl, or C₆-C₂₀ aryl;

Z¹ is selected from —(C₁-C₁₂ alkylene)-, —(C₂-C₈ alkenylene)-, —(C₂-C₈alkynylene)-, and —(CH₂CH₂O)_(n)—,

m is 0 or 1;

n is 1 to 6;

p is an integer from 1 to 8; and

alkylene, alkenylene, alkynylene, alkyl, and aryl are optionallysubstituted with one or more groups selected from F, Cl, Br, N(CH₃)₂,NO₂, and OCH₃.

Another aspect of the invention is a pharmaceutical compositioncomprising an antibody-drug conjugate compound of Formula II, and apharmaceutically acceptable diluent, carrier or excipient.

Another aspect of the invention is the use of an antibody-drug conjugatecompound of Formula II in the manufacture of a medicament for thetreatment of cancer in a mammal.

Another aspect of the invention is a method of treating cancer byadministering to a patient a pharmaceutical composition comprising anantibody-drug conjugate compound of Formula II.

Another aspect of the invention is a method of making an antibody-drugconjugate compound of Formula II.

Another aspect of the invention is an article of manufacture comprisinga pharmaceutical composition comprising an antibody-drug conjugatecompound of Formula II, a container, and a package insert or labelindicating that the pharmaceutical composition can be used to treatcancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the efficacy of antibody-drug conjugates in a plot ofSK-BR-3 in vitro cell viability at 5 days versus concentrations (μg/ml)of Thio Hu anti-Her2 7C2 HC A118C-(LD-57) 114, Thio Hu anti-Her2 7C2 LCK149C-(LD-57) 109, and Thio Hu anti-CD33 15G15.3 LC K149C-(LD-57) 110.

FIG. 2 shows the efficacy of antibody-drug conjugates in a plot ofSK-BR-3 in vitro cell viability at 5 days versus concentrations (μg/ml)of Thio Hu anti-Her2 7C2 LC K149C-(LD-59) 116, Thio Hu anti-Her2 7C2 LCK149C-(LD-57) 109, and Thio Hu Anti-CD33 15G15.3 LC K149C-(LD-59) 115.

FIG. 3 shows the efficacy of antibody-drug conjugates in a plot of thein vivo fitted tumor volume change over time in MMTV-HER2 Fo5 transgenicmammary tumors inoculated into the mammary fat pad of CRL nu/nu miceafter dosing once IV with: Vehicle: Histidine Buffer #8: 20 mM HistidineAcetate, pH 5.5, 240 mM Sucrose, 0.02% PS 20, Thio Hu Anti-Her2 7C2 HCA118C-(LD-51) 107, and Thio Hu Anti-Her2 7C2 LC K149C-(LD-51) 108. ADCwere dosed at 3 mg/kg one time IV at day 0.

FIG. 4 shows the efficacy of antibody-drug conjugates in a plot of thein vivo fitted tumor volume change over time in KPL4 tumor model in scidbeige mice inoculated in the thoracic mammary fat pad at a volume of 0.2ml. When tumors reached a mean tumor volume of 100-250 mm3, they weregrouped into 9 groups of 8-10 mice each. A single treatment wasadministered intravenously via the tail vein on Day 0 as follows: (01)Vehicle: Histidine Buffer #8: 20 mM Histidine Acetate, pH 5.5, 240 mMSucrose, 0.02% PS 20, (02) Thio Hu anti-Her2 7C2 LCK149C-(ethylmaleimide) 0.3 mg/kg 138, (03) Thio Hu anti-Her2 7C2 LCK149C-(ethylmaleimide) 1 mg/kg 138, (04) Thio Hu anti-Her2 7C2 LCK149C-(ethylmaleimide) 3 mg/kg 138, (05) Thio Hu anti-Her2 7C2 LCK149C-(LD-57) 0.3 mg/kg 109, (06) Thio Hu anti-Her2 7C2 LC K149C-(LD-57)1 mg/kg 109, (07) Thio Hu anti-Her2 7C2 LC K149C-(LD-57)3 mg/kg 109,(08) Thio Hu Anti-CD33 15G15.3 LC K149C-(ethylmaleimide) 3 mg/kg 139,and (09) Thio Hu anti-CD33 15G15.3 LC K149C-(LD-57) 3 mg/kg 110.

FIG. 5 shows the efficacy of antibody-drug conjugates in a plot of thein vivo fitted tumor volume change over time in MMTV-HER2 Fo5 transgenicmammary tumors inoculated into the mammary fat pad of CRL nu/nu miceafter dosing once IV with: (01) Vehicle: Histidine Buffer #8: 20 mMHistidine Acetate, pH 5.5, 240 mM Sucrose, 0.02% PS 20, (02) Thio Huanti-Her2 7C2 LC K149C-(LD-57) 1 mg/kg 109, (03) Thio Hu anti-Her2 7C2LC K149C-(LD-57) 3 mg/kg 109, (04) Thio Hu anti-CD33 15G15.3 LCK149C-(LD-57) 1 mg/kg 110, (05) Thio Hu anti-CD33 15G15.3 LCK149C-(LD-57) 3 mg/kg 110, (06) trastuzumab emtansine 3 mg/kg 141, and(07) trastuzumab emtansine 10 mg/kg 141.

FIG. 6 shows the efficacy of antibody-drug conjugates in a plot ofBJAB.luc in vitro cell viability at 5 days versus concentrations (μg/ml)of Thio Hu Anti-Napi3b 10H1.11.4B LC K149C-(LD-57) 121, Thio Huanti-CD22 10F4v3 LC K149C-(LD-57) 120, Thio Hu Anti-Napi3b 10H1.11.4B LCK149C-(LD-59) 123, Thio Hu anti-CD22 10F4v3 LC K149C-(LD-59) 122, ThioHu Anti-Napi3b 10H1.11.4B LC K149C-(LD-58) 125, Thio Hu anti-CD22 10F4v3LC K149C-(LD-58) 124, Thio Hu Anti-Napi3b 10H1.11.4B LC K149C-(LD-60)127, and Thio Hu anti-CD22 10F4v3 LC K149C-(LD-60) 126.

FIG. 7 shows the efficacy of antibody-drug conjugates in a plot ofWSU-DLCL2 in vitro cell viability at 5 days versus concentrations(μg/ml) of Thio Hu Anti-Napi3b 10H1.11.4B LC K149C-(LD-57) 121, Thio Huanti-CD22 10F4v3 LC K149C-(LD-57) 120, Thio Hu Anti-Napi3b 10H1.11.4B LCK149C-(LD-59) 123, Thio Hu anti-CD22 10F4v3 LC K149C-(LD-59) 122, ThioHu Anti-Napi3b 10H1.11.4B LC K149C-(LD-58) 125, Thio Hu anti-CD22 10F4v3LC K149C-(LD-58) 124, Thio Hu Anti-Napi3b 10H1.11.4B LC K149C-(LD-60)127, and Thio Hu anti-CD22 10F4v3 LC K149C-(LD-60) 126.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

Reference will now be made in detail to certain embodiments of theinvention, examples of which are illustrated in the accompanyingstructures and formulas. While the invention will be described inconjunction with the illustrated embodiments, it will be understood thatthey are not intended to limit the invention to those embodiments. Onthe contrary, the invention is intended to cover all alternatives,modifications, and equivalents, which may be included within the scopeof the present invention as defined by the claims.

One skilled in the art will recognize many methods and materials similaror equivalent to those described herein, which could be used in thepractice of the present invention. The present invention is in no waylimited to the methods and materials described.

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs, and are consistent with:Singleton et al (1994) Dictionary of Microbiology and Molecular Biology,2nd Ed., J. Wiley & Sons, New York, N.Y.; and Janeway, C., Travers, P.,Walport, M., Shlomchik (2001) Immunobiology, 5th Ed., GarlandPublishing, New York.

Definitions

Unless stated otherwise, the following terms and phrases as used hereinare intended to have the following meanings:

When trade names are used herein, applicants intend to independentlyinclude the trade name product formulation, the generic drug, and theactive pharmaceutical ingredient(s) of the trade name product.

The term “antibody” herein is used in the broadest sense andspecifically covers monoclonal antibodies, polyclonal antibodies,dimers, multimers, multi specific antibodies (e.g., bispecificantibodies), and antibody fragments, so long as they exhibit the desiredbiological activity (Miller et al (2003) Jour. of Immunology170:4854-4861). Antibodies may be murine, human, humanized, chimeric, orderived from other species. An antibody is a protein generated by theimmune system that is capable of recognizing and binding to a specificantigen. (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) ImmunoBiology, 5th Ed., Garland Publishing, New York). A target antigengenerally has numerous binding sites, also called epitopes, recognizedby CDRs on multiple antibodies. Each antibody that specifically binds toa different epitope has a different structure. Thus, one antigen mayhave more than one corresponding antibody. An antibody includes afull-length immunoglobulin molecule or an immunologically active portionof a full-length immunoglobulin molecule, i.e., a molecule that containsan antigen binding site that immunospecifically binds an antigen of atarget of interest or part thereof, such targets including but notlimited to, cancer cell or cells that produce autoimmune antibodiesassociated with an autoimmune disease. The immunoglobulin disclosedherein can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class(e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass ofimmunoglobulin molecule. The immunoglobulins can be derived from anyspecies. In one aspect, however, the immunoglobulin is of human, murine,or rabbit origin.

“Antibody fragments” comprise a portion of a full length antibody,generally the antigen binding or variable region thereof. Examples ofantibody fragments include Fab, Fab′, F(ab′)2, and Fv fragments;diabodies; linear antibodies; minibodies (Olafsen et al (2004) ProteinEng. Design & Sel. 17(4):315-323), fragments produced by a Fabexpression library, anti-idiotypic (anti-Id) antibodies, CDR(complementary determining region), and epitope-binding fragments of anyof the above which immunospecifically bind to cancer cell antigens,viral antigens or microbial antigens, single-chain antibody molecules;and multispecific antibodies formed from antibody fragments.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to polyclonalantibody preparations which include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody isdirected against a single determinant on the antigen. In addition totheir specificity, the monoclonal antibodies are advantageous in thatthey may be synthesized uncontaminated by other antibodies. The modifier“monoclonal” indicates the character of the antibody as being obtainedfrom a substantially homogeneous population of antibodies, and is not tobe construed as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by the hybridoma method firstdescribed by Kohler et al (1975) Nature, 256:495, or may be made byrecombinant DNA methods (see for example: U.S. Pat. No. 4,816,567; U.S.Pat. No. 5,807,715). Monoclonal antibodies may also be isolated fromphage antibody libraries using the techniques described in Clackson etal (1991) Nature, 352:624-628; Marks et al (1991) J. Mol. Biol.,222:581-597.

Monoclonal antibodies herein specifically include “chimeric” antibodiesin which a portion of the heavy and/or light chain is identical with orhomologous to corresponding sequences in antibodies derived from aparticular species or belonging to a particular antibody class orsubclass, while the remainder of the chain(s) is identical with orhomologous to corresponding sequences in antibodies derived from anotherspecies or belonging to another antibody class or subclass, as well asfragments of such antibodies, so long as they exhibit the desiredbiological activity (U.S. Pat. No. 4,816,567; and Morrison et al (1984)Proc. Natl. Acad. Sci. USA, 81:6851-6855). Chimeric antibodies ofinterest herein include “primatized” antibodies comprising variabledomain antigen-binding sequences derived from a non-human primate (e.g.,Old World Monkey, Ape, etc.) and human constant region sequences.

An “intact antibody” herein is one comprising a VL and VH domains, aswell as a light chain constant domain (CL) and heavy chain constantdomains, CH1, CH2 and CH3. The constant domains may be native sequenceconstant domains (e.g., human native sequence constant domains) or aminoacid sequence variant thereof. The intact antibody may have one or more“effector functions” which refer to those biological activitiesattributable to the Fc constant region (a native sequence Fc region oramino acid sequence variant Fc region) of an antibody. Examples ofantibody effector functions include C1q binding; complement dependentcytotoxicity; Fc receptor binding; antibody-dependent cell-mediatedcytotoxicity (ADCC); phagocytosis; and down regulation of cell surfacereceptors such as B cell receptor and BCR.

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain that contains at least a portion of theconstant region. The term includes native sequence Fc regions andvariant Fc regions. In one embodiment, a human IgG heavy chain Fc regionextends from Cys226, or from Pro230, to the carboxyl-terminus of theheavy chain. However, the C-terminal lysine (Lys447) of the Fc regionmay or may not be present. Unless otherwise specified herein, numberingof amino acid residues in the Fc region or constant region is accordingto the EU numbering system, also called the EU index, as described inKabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.,1991.

“Framework” or “FR” refers to variable domain residues other thanhypervariable region (HVR) residues. The FR of a variable domaingenerally consists of four FR domains: FR1, FR2, FR3, and FR4.Accordingly, the HVR and FR sequences generally appear in the followingsequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

Depending on the amino acid sequence of the constant domain of theirheavy chains, intact antibodies can be assigned to different “classes.”There are five major classes of intact immunoglobulin antibodies: IgA,IgD, IgE, IgG, and IgM, and several of these may be further divided into“subclasses” (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.The heavy-chain constant domains that correspond to the differentclasses of antibodies are called α, δ, ε, γ, and μ, respectively. Thesubunit structures and three-dimensional configurations of differentclasses of immunoglobulins are well known. Ig forms includehinge-modifications or hingeless forms (Roux et al (1998) J. Immunol.161:4083-4090; Lund et al (2000) Eur. J. Biochem. 267:7246-7256; US2005/0048572; US 2004/0229310).

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human or a human cellor derived from a non-human source that utilizes human antibodyrepertoires or other human antibody-encoding sequences. This definitionof a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues.

A “human consensus framework” is a framework which represents the mostcommonly occurring amino acid residues in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat et al., Sequences of Proteins of Immunological Interest, FifthEdition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3. In oneembodiment, for the VL, the subgroup is subgroup kappa I as in Kabat etal., supra. In one embodiment, for the VH, the subgroup is subgroup IIIas in Kabat et al., supra.

A “humanized” antibody refers to a chimeric antibody comprising aminoacid residues from non-human HVRs and amino acid residues from humanFRs. In certain embodiments, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the HVRs (e.g., CDRs) correspond tothose of a non-human antibody, and all or substantially all of the FRscorrespond to those of a human antibody. A humanized antibody optionallymay comprise at least a portion of an antibody constant region derivedfrom a human antibody. A “humanized form” of an antibody, e.g., anon-human antibody, refers to an antibody that has undergonehumanization.

The term “hypervariable region” or “HVR,” as used herein, refers to eachof the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops (“hypervariable loops”).Generally, native four-chain antibodies comprise six HVRs; three in theVH (H1, H2, H3), and three in the VL (L1, L2, L3). HVRs generallycomprise amino acid residues from the hypervariable loops and/or fromthe “complementarity determining regions” (CDRs), the latter being ofhighest sequence variability and/or involved in antigen recognition.Exemplary hypervariable loops occur at amino acid residues 26-32 (L1),50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3).(Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987).) Exemplary CDRs(CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) occur at amino acidresidues 24-34 of L1, 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 ofH2, and 95-102 of H3. (Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991).) With the exception of CDR1in VH, CDRs generally comprise the amino acid residues that form thehypervariable loops. CDRs also comprise “specificity determiningresidues,” or “SDRs,” which are residues that contact antigen. SDRs arecontained within regions of the CDRs called abbreviated-CDRs, or a-CDRs.Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, anda-CDR-H3) occur at amino acid residues 31-34 of L1, 50-55 of L2, 89-96of L3, 31-35B of H1, 50-58 of H2, and 95-102 of H3. (See Almagro andFransson, Front. Biosci. 13:1619-1633 (2008).) Unless otherwiseindicated, HVR residues and other residues in the variable domain (e.g.,FR residues) are numbered herein according to Kabat et al., supra.

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable domains of the heavy chain and lightchain (VH and VL, respectively) of a native antibody generally havesimilar structures, with each domain comprising four conserved frameworkregions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindtet al. Kuby Immunology, 6^(th) ed., W.H. Freeman and Co., page 91(2007).) A single VH or VL domain may be sufficient to conferantigen-binding specificity. Furthermore, antibodies that bind aparticular antigen may be isolated using a VH or VL domain from anantibody that binds the antigen to screen a library of complementary VLor VH domains, respectively. See, e.g., Portolano et al., J. Immunol.150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

The term “vector,” as used herein, refers to a nucleic acid moleculecapable of propagating another nucleic acid to which it is linked. Theterm includes the vector as a self-replicating nucleic acid structure aswell as the vector incorporated into the genome of a host cell intowhich it has been introduced. Certain vectors are capable of directingthe expression of nucleic acids to which they are operatively linked.Such vectors are referred to herein as “expression vectors.”

A “free cysteine amino acid” refers to a cysteine amino acid residuewhich has been engineered into a parent antibody, has a thiol functionalgroup (—SH), and is not paired as an intramolecular or intermoleculardisulfide bridge.

“Linker”, “Linker Unit”, or “link” means a chemical moiety comprising achain of atoms that covalently attaches an antibody to a drug moiety. Invarious embodiments, a linker is a divalent radical, specified as L.

When indicating the number of substituents, the term “one or more”refers to the range from one substituent to the highest possible numberof substitution, i.e. replacement of one hydrogen up to replacement ofall hydrogens by substituents. The term “substituent” denotes an atom ora group of atoms replacing a hydrogen atom on the parent molecule. Theterm “substituted” denotes that a specified group bears one or moresubstituents. Where any group may carry multiple substituents and avariety of possible substituents is provided, the substituents areindependently selected and need not to be the same. The term“unsubstituted” means that the specified group bears no substituents.The term “optionally substituted” means that the specified group isunsubstituted or substituted by one or more substituents, independentlychosen from the group of possible substituents. When indicating thenumber of substituents, the term “one or more” means from onesubstituent to the highest possible number of substitution, i.e.replacement of one hydrogen up to replacement of all hydrogens bysubstituents.

The term “alkyl” as used herein refers to a saturated linear orbranched-chain monovalent hydrocarbon radical of any length from one totwelve carbon atoms (C₁-C₁₂), wherein the alkyl radical may beoptionally substituted independently with one or more substituentsdescribed below. In another embodiment, an alkyl radical is one to eightcarbon atoms (C₁-C₈), or one to six carbon atoms (C₁-C₆). Examples ofalkyl groups include, but are not limited to, methyl (Me, —CH₃), ethyl(Et, —CH₂CH₃), 1-propyl (n-Pr, n-propyl, —CH₂CH₂CH₃), 2-propyl (i-Pr,i-propyl, —CH(CH₃)₂), 1-butyl (n-Bu, n-butyl, —CH₂CH₂CH₂CH₃),2-methyl-1-propyl (i-Bu, i-butyl, —CH₂CH(CH₃)₂), 2-butyl (s-Bu, s-butyl,—CH(CH₃)CH₂CH₃), 2-methyl-2-propyl (t-Bu, t-butyl, —C(CH₃)₃), 1-pentyl(n-pentyl, —CH₂CH₂CH₂CH₂CH₃), 2-pentyl (—CH(CH₃)CH₂CH₂CH₃), 3-pentyl(—CH(CH₂CH₃)₂), 2-methyl-2-butyl (—C(CH₃)₂CH₂CH₃), 3-methyl-2-butyl(—CH(CH₃)CH(CH₃)₂), 3-methyl-1-butyl (—CH₂CH₂CH(CH₃)₂), 2-methyl-1-butyl(—CH₂CH(CH₃)CH₂CH₃), 1-hexyl (—CH₂CH₂CH₂CH₂CH₂CH₃), 2-hexyl(—CH(CH₃)CH₂CH₂CH₂CH₃), 3-hexyl (—CH(CH₂CH₃)(CH₂CH₂CH₃)),2-methyl-2-pentyl (—C(CH₃)₂CH₂CH₂CH₃), 3-methyl-2-pentyl(—CH(CH₃)CH(CH₃)CH₂CH₃), 4-methyl-2-pentyl (—CH(CH₃)CH₂CH(CH₃)₂),3-methyl-3-pentyl (—C(CH₃)(CH₂CH₃)₂), 2-methyl-3-pentyl(—CH(CH₂CH₃)CH(CH₃)₂), 2,3-dimethyl-2-butyl (—C(CH₃)₂CH(CH₃)₂),3,3-dimethyl-2-butyl (—CH(CH₃)C(CH₃)₃, 1-heptyl, 1-octyl, and the like.

The term “alkylene” as used herein refers to a saturated linear orbranched-chain divalent hydrocarbon radical of any length from one totwelve carbon atoms (C₁-C₁₂), wherein the alkylene radical may beoptionally substituted independently with one or more substituentsdescribed below. In another embodiment, an alkylene radical is one toeight carbon atoms (C₁-C₈), or one to six carbon atoms (C₁-C₆). Examplesof alkylene groups include, but are not limited to, methylene (—CH₂—),ethylene (—CH₂CH₂—), n-propylene (—CH₂CH₂CH₂—), i-propylene(—CH(CH₃)CH₂—), i-butylene (—C(CH₃)₂CH₂—) and the like.

The term “alkenyl” refers to linear or branched-chain monovalenthydrocarbon radical of any length from two to eight carbon atoms (C₂-C₈)with at least one site of unsaturation, i.e., a carbon-carbon, sp²double bond, wherein the alkenyl radical may be optionally substitutedindependently with one or more substituents described herein, andincludes radicals having “cis” and “trans” orientations, oralternatively, “E” and “Z” orientations. Examples include, but are notlimited to, ethylenyl or vinyl (—CH═CH₂), allyl (—CH₂CH═CH₂), crotyl(—CH(CH₃)CH═CH₂), and the like.

The term “alkenylene” refers to linear or branched-chain divalenthydrocarbon radical of any length from two to eight carbon atoms (C₂-C₈)with at least one site of unsaturation, i.e., a carbon-carbon, sp²double bond, wherein the alkenylene radical may be optionallysubstituted independently with one or more substituents describedherein, and includes radicals having “cis” and “trans” orientations, oralternatively, “E” and “Z” orientations. Examples include, but are notlimited to, ethylenylene or vinylene (—CH═CH—), allylene (—CH₂CH═CH—),crotylene (—CH(CH₃)CH═CH—), and the like.

The term “alkynyl” refers to a linear or branched monovalent hydrocarbonradical of any length from two to eight carbon atoms (C₂-C₈) with atleast one site of unsaturation, i.e., a carbon-carbon, sp triple bond,wherein the alkynyl radical may be optionally substituted independentlywith one or more substituents described herein. Examples include, butare not limited to, ethynyl (—C≡CH), propynyl (propargyl, —CH₂C≡CH), andthe like.

The term “alkynylene” refers to a linear or branched divalenthydrocarbon radical of any length from two to eight carbon atoms (C₂-C₈)with at least one site of unsaturation, i.e., a carbon-carbon, sp triplebond, wherein the alkynylene radical may be optionally substitutedindependently with one or more substituents described herein. Examplesinclude, but are not limited to, ethynylene propynylene (propargylene,—CH₂C≡C—), 2-butynylene (—CH(CH₃)C≡C—), and the like.

The terms “carbocycle”, “carbocyclyl”, “carbocyclic ring” and“cycloalkyl” refer to a monovalent non-aromatic, saturated or partiallyunsaturated ring having 3 to 12 carbon atoms (C₃-C₁₂) as a monocyclicring or 7 to 12 carbon atoms as a bicyclic ring. Bicyclic carbocycleshaving 7 to 12 atoms can be arranged, for example, as a bicyclo[4,5],[5,5], [5,6] or [6,6] system, and bicyclic carbocycles having 9 or 10ring atoms can be arranged as a bicyclo[5,6] or [6,6] system, or asbridged systems such as bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane andbicyclo[3.2.2]nonane. Spiro moieties are also included within the scopeof this definition. Examples of monocyclic carbocycles include, but arenot limited to, cyclopropyl, cyclobutyl, cyclopentyl,1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl,1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl,cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl,cycloundecyl, cyclododecyl, and the like. Carbocyclyl groups areoptionally substituted independently with one or more substituentsdescribed herein.

“Aryl” means a monovalent aromatic hydrocarbon radical of 6-20 carbonatoms (C₆-C₂₀) derived by the removal of one hydrogen atom from a singlecarbon atom of a parent aromatic ring system. Some aryl groups arerepresented in the exemplary structures as “Ar”. Aryl includes bicyclicradicals comprising an aromatic ring fused to a saturated, partiallyunsaturated ring, or aromatic carbocyclic ring. Typical aryl groupsinclude, but are not limited to, radicals derived from benzene (phenyl),substituted benzenes, naphthalene, anthracene, biphenyl, indenyl,indanyl, 1,2-dihydronaphthalene, 1,2,3,4-tetrahydronaphthyl, and thelike. Aryl groups are optionally substituted independently with one ormore substituents described herein.

“Arylene” means a divalent aromatic hydrocarbon radical of 6-20 carbonatoms (C₆-C₂₀) derived by the removal of two hydrogen atom from a twocarbon atoms of a parent aromatic ring system. Some arylene groups arerepresented in the exemplary structures as “Ar”. Arylene includesbicyclic radicals comprising an aromatic ring fused to a saturated,partially unsaturated ring, or aromatic carbocyclic ring. Typicalarylene groups include, but are not limited to, radicals derived frombenzene (phenylene), substituted benzenes, naphthalene, anthracene,biphenylene, indenylene, indanylene, 1,2-dihydronaphthalene,1,2,3,4-tetrahydronaphthyl, and the like. Arylene groups are optionallysubstituted with one or more substituents described herein.

The terms “heterocycle,” “heterocyclyl” and “heterocyclic ring” are usedinterchangeably herein and refer to a saturated or a partiallyunsaturated (i.e., having one or more double and/or triple bonds withinthe ring) carbocyclic radical of 3 to about 20 ring atoms in which atleast one ring atom is a heteroatom selected from nitrogen, oxygen,phosphorus and sulfur, the remaining ring atoms being C, where one ormore ring atoms is optionally substituted independently with one or moresubstituents described below. A heterocycle may be a monocycle having 3to 7 ring members (2 to 6 carbon atoms and 1 to 4 heteroatoms selectedfrom N, O, P, and S) or a bicycle having 7 to 10 ring members (4 to 9carbon atoms and 1 to 6 heteroatoms selected from N, O, P, and S), forexample: a bicyclo[4,5], [5,5], [5,6], or [6,6] system. Heterocycles aredescribed in Paquette, Leo A.; “Principles of Modern HeterocyclicChemistry” (W. A. Benjamin, New York, 1968), particularly Chapters 1, 3,4, 6, 7, and 9; “The Chemistry of Heterocyclic Compounds, A series ofMonographs” (John Wiley & Sons, New York, 1950 to present), inparticular Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc. (1960)82:5566. “Heterocyclyl” also includes radicals where heterocycleradicals are fused with a saturated, partially unsaturated ring, oraromatic carbocyclic or heterocyclic ring. Examples of heterocyclicrings include, but are not limited to, morpholin-4-yl, piperidin-1-yl,piperazinyl, piperazin-4-yl-2-one, piperazin-4-yl-3-one,pyrrolidin-1-yl, thiomorpholin-4-yl, S-dioxothiomorpholin-4-yl,azocan-1-yl, azetidin-1-yl, octahydropyrido[1,2-a]pyrazin-2-yl,[1,4]diazepan-1-yl, pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl,tetrahydrothienyl, tetrahydropyranyl, dihydropyranyl,tetrahydrothiopyranyl, piperidino, morpholino, thiomorpholino,thioxanyl, piperazinyl, homopiperazinyl, azetidinyl, oxetanyl,thietanyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl,thiazepinyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl,4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl, dithianyl,dithiolanyl, dihydropyranyl, dihydrothienyl, dihydrofuranyl,pyrazolidinylimidazolinyl, imidazolidinyl, 3-azabicyco[3.1.0]hexanyl,3-azabicyclo[4.1.0]heptanyl, azabicyclo[2.2.2]hexanyl, 3H-indolylquinolizinyl and N-pyridyl ureas. Spiro moieties are also includedwithin the scope of this definition. Examples of a heterocyclic groupwherein 2 ring atoms are substituted with oxo (═O) moieties arepyrimidinonyl and 1,1-dioxo-thiomorpholinyl. The heterocycle groupsherein are optionally substituted independently with one or moresubstituents described herein.

The term “heteroaryl” refers to a monovalent aromatic radical of 5-, 6-,or 7-membered rings, and includes fused ring systems (at least one ofwhich is aromatic) of 5-20 atoms, containing one or more heteroatomsindependently selected from nitrogen, oxygen, and sulfur. Examples ofheteroaryl groups are pyridinyl (including, for example,2-hydroxypyridinyl), imidazolyl, imidazopyridinyl,1-methyl-1H-benzo[d]imidazole, [1,2,4]triazolo[1,5-a]pyridine,pyrimidinyl (including, for example, 4-hydroxypyrimidinyl), pyrazolyl,triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl,oxadiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolinyl,isoquinolinyl, tetrahydroisoquinolinyl, indolyl, benzimidazolyl,benzofuranyl, cinnolinyl, indazolyl, indolizinyl, phthalazinyl,pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl,thiadiazolyl, thiadiazolyl, furazanyl, benzofurazanyl, benzothiophenyl,benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl,naphthyridinyl, and furopyridinyl. Heteroaryl groups are optionallysubstituted independently with one or more substituents describedherein.

The heterocycle or heteroaryl groups may be carbon (carbon-linked), ornitrogen (nitrogen-linked) bonded where such is possible. By way ofexample and not limitation, carbon bonded heterocycles or heteroarylsare bonded at position 2, 3, 4, 5, or 6 of a pyridine, position 3, 4, 5,or 6 of a pyridazine, position 2, 4, 5, or 6 of a pyrimidine, position2, 3, 5, or 6 of a pyrazine, position 2, 3, 4, or 5 of a furan,tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrrole,position 2, 4, or 5 of an oxazole, imidazole or thiazole, position 3, 4,or 5 of an isoxazole, pyrazole, or isothiazole, position 2 or 3 of anaziridine, position 2, 3, or 4 of an azetidine, position 2, 3, 4, 5, 6,7, or 8 of a quinoline or position 1, 3, 4, 5, 6, 7, or 8 of anisoquinoline.

By way of example and not limitation, nitrogen bonded heterocycles orheteroaryls are bonded at position 1 of an aziridine, azetidine,pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole,imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline,2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline,1H-indazole, position 2 of a isoindole, or isoindoline, position 4 of amorpholine, and position 9 of a carbazole, or (3-carboline.

The term “chiral” refers to molecules which have the property ofnon-superimposability of the mirror image partner, while the term“achiral” refers to molecules which are superimposable on their mirrorimage partner.

The term “stereoisomers” refers to compounds which have identicalchemical constitution, but differ with regard to the arrangement of theatoms or groups in space.

“Diastereomer” refers to a stereoisomer with two or more centers ofchirality and whose molecules are not mirror images of one another.Diastereomers have different physical properties, e.g. melting points,boiling points, spectral properties, and reactivities. Mixtures ofdiastereomers may separate under high resolution analytical proceduressuch as electrophoresis and chromatography.

“Enantiomers” refer to two stereoisomers of a compound which arenon-superimposable mirror images of one another.

Stereochemical definitions and conventions used herein generally followS. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984)McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S.,Stereochemistry of Organic Compounds (1994) John Wiley & Sons, Inc., NewYork. Many organic compounds exist in optically active forms, i.e., theyhave the ability to rotate the plane of plane-polarized light. Indescribing an optically active compound, the prefixes D and L, or R andS, are used to denote the absolute configuration of the molecule aboutits chiral center(s). The prefixes d and 1 or (+) and (−) are employedto designate the sign of rotation of plane-polarized light by thecompound, with (−) or 1 meaning that the compound is levorotatory. Acompound prefixed with (+) or d is dextrorotatory. For a given chemicalstructure, these stereoisomers are identical except that they are mirrorimages of one another. A specific stereoisomer may also be referred toas an enantiomer, and a mixture of such isomers is often called anenantiomeric mixture. A 50:50 mixture of enantiomers is referred to as aracemic mixture or a racemate, which may occur where there has been nostereoselection or stereospecificity in a chemical reaction or process.The terms “racemic mixture” and “racemate” refer to an equimolar mixtureof two enantiomeric species, devoid of optical activity.

The phrase “pharmaceutically acceptable salt,” as used herein, refers topharmaceutically acceptable organic or inorganic salts of anantibody-drug conjugate (ADC). Exemplary salts include, but are notlimited, to sulfate, citrate, acetate, oxalate, chloride, bromide,iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate,lactate, salicylate, acid citrate, tartrate, oleate, tannate,pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate,fumarate, gluconate, glucuronate, saccharate, formate, benzoate,glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate,p-toluenesulfonate, and pamoate (i.e.,1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts. A pharmaceuticallyacceptable salt may involve the inclusion of another molecule such as anacetate ion, a succinate ion or other counterion. The counterion may beany organic or inorganic moiety that stabilizes the charge on the parentcompound. Furthermore, a pharmaceutically acceptable salt may have morethan one charged atom in its structure. Instances where multiple chargedatoms are part of the pharmaceutically acceptable salt can have multiplecounter ions. Hence, a pharmaceutically acceptable salt can have one ormore charged atoms and/or one or more counterion.

The following abbreviations are used herein and have the indicateddefinitions: BME is beta-mercaptoethanol, Boc is N-(t-butoxycarbonyl),cit is citrulline (2-amino-5-ureido pentanoic acid), DCC is1,3-dicyclohexylcarbodiimide, DCM is dichloromethane, DEA isdiethylamine, DEAD is diethylazodicarboxylate, DEPC isdiethylphosphorylcyanidate, DIAD is diisopropylazodicarboxylate, DIEA isN,N-diisopropylethylamine, DMA is dimethylacetamide, DMAP is4-dimethylaminopyridine, DME is ethyleneglycol dimethyl ether (or1,2-dimethoxyethane), DMF is N,N-dimethylformamide, DMSO isdimethylsulfoxide, DTT is dithiothreitol, EDCI is1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, EEDQ is2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, ES-MS is electrospraymass spectrometry, EtOAc is ethyl acetate, Fmoc isN-(9-fluorenylmethoxycarbonyl), gly is glycine, HATU isO-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate, HOBt is 1-hydroxybenzotriazole, HPLC is highpressure liquid chromatography, ile is isoleucine, lys is lysine, MeCN(CH₃CN) is acetonitrile, MeOH is methanol, Mtr is 4-anisyldiphenylmethyl(or 4-methoxytrityl), NHS is N-hydroxysuccinimide, PBS isphosphate-buffered saline (pH 7), PEG is polyethylene glycol or a unitof ethylene glycol (—OCH₂CH₂—), Ph is phenyl, Pnp is p-nitrophenyl, MCis 6-maleimidocaproyl, phe is L-phenylalanine, PyBrop is bromotris-pyrrolidino phosphonium hexafluorophosphate, SEC is size-exclusionchromatography, Su is succinimide, TFA is trifluoroacetic acid, TLC isthin layer chromatography, UV is ultraviolet, and val is valine.

Cysteine Engineered Antibodies

The compounds of the invention include antibody-drug conjugatescomprising cysteine engineered antibodies where one or more amino acidsof a wild-type or parent antibody are replaced with a cysteine aminoacid (THIOMAB™). Any form of antibody may be so engineered, i.e.mutated. For example, a parent Fab antibody fragment may be engineeredto form a cysteine engineered Fab. Similarly, a parent monoclonalantibody may be engineered to form a THIOMAB™. It should be noted that asingle site mutation yields a single engineered cysteine residue in aFab antibody fragment, while a single site mutation yields twoengineered cysteine residues in a full length THIOMAB™, due to thedimeric nature of the IgG antibody. Mutants with replaced (“engineered”)cysteine (Cys) residues are evaluated for the reactivity of the newlyintroduced, engineered cysteine thiol groups. The thiol reactivity valueis a relative, numerical term in the range of 0 to 1.0 and can bemeasured for any cysteine engineered antibody. Thiol reactivity valuesof cysteine engineered antibodies of the invention are in the ranges of0.6 to 1.0; 0.7 to 1.0; or 0.8 to 1.0.

Cysteine amino acids may be engineered at reactive sites in the heavychain (HC) or light chain (LC) of an antibody and which do not formintrachain or intermolecular disulfide linkages (Junutula, et al., 2008bNature Biotech., 26(8):925-932; Dornan et al (2009) Blood114(13):2721-2729; U.S. Pat. No. 7,521,541; U.S. Pat. No. 7,723,485;WO2009/052249, Shen et al (2012) Nature Biotech., 30(2):184-191;Junutula et al (2008) Jour of Immun. Methods 332:41-52). The engineeredcysteine thiols may react with linker reagents or the linker-drugintermediates of the present invention which have thiol-reactive,electrophilic pyridyl disulfide groups to form ADC with cysteineengineered antibodies (THIOMAB™) and the drug (D) moiety. The locationof the drug moiety can thus be designed, controlled, and known. The drugloading can be controlled since the engineered cysteine thiol groupstypically react with thiol-reactive linker reagents or linker-drugintermediates in high yield. Engineering an antibody to introduce acysteine amino acid by substitution at a single site on the heavy orlight chain gives two new cysteines on the symmetrical antibody. A drugloading near 2 can be achieved and near homogeneity of the conjugationproduct ADC.

Cysteine engineered antibodies of the invention preferably retain theantigen binding capability of their wild type, parent antibodycounterparts. Thus, cysteine engineered antibodies are capable ofbinding, preferably specifically, to antigens. Such antigens include,for example, tumor-associated antigens (TAA), cell surface receptorproteins and other cell surface molecules, transmembrane proteins,signaling proteins, cell survival regulatory factors, cell proliferationregulatory factors, molecules associated with (for e.g., known orsuspected to contribute functionally to) tissue development ordifferentiation, lymphokines, cytokines, molecules involved in cellcycle regulation, molecules involved in vasculogenesis and moleculesassociated with (for e.g., known or suspected to contribute functionallyto) angiogenesis. The tumor-associated antigen may be a clusterdifferentiation factor (i.e., a CD protein). An antigen to which acysteine engineered antibody is capable of binding may be a member of asubset of one of the above-mentioned categories, wherein the othersubset(s) of said category comprise other molecules/antigens that have adistinct characteristic (with respect to the antigen of interest).

Cysteine engineered antibodies are prepared for conjugation withlinker-drug intermediates by reduction and reoxidation of intrachaindisulfide groups (Example 19).

Cysteine engineered antibodies which may be useful in the antibody-drugconjugates of the invention in the treatment of cancer include, but arenot limited to, antibodies against cell surface receptors andtumor-associated antigens (TAA). Tumor-associated antigens are known inthe art, and can be prepared for use in generating antibodies usingmethods and information which are well known in the art. In attempts todiscover effective cellular targets for cancer diagnosis and therapy,researchers have sought to identify transmembrane or otherwisetumor-associated polypeptides that are specifically expressed on thesurface of one or more particular type(s) of cancer cell as compared toon one or more normal non-cancerous cell(s). Often, suchtumor-associated polypeptides are more abundantly expressed on thesurface of the cancer cells as compared to on the surface of thenon-cancerous cells. The identification of such tumor-associated cellsurface antigen polypeptides has given rise to the ability tospecifically target cancer cells for destruction via antibody-basedtherapies.

Examples of tumor-associated antigens TAA include, but are not limitedto, TAA (1)-(51) listed below. For convenience, information relating tothese antigens, all of which are known in the art, is listed below andincludes names, alternative names, Genbank accession numbers and primaryreference(s), following nucleic acid and protein sequence identificationconventions of the National Center for Biotechnology Information (NCBI).Nucleic acid and protein sequences corresponding to TAA (1)-(53) areavailable in public databases such as GenBank. Tumor-associated antigenstargeted by antibodies include all amino acid sequence variants andisoforms possessing at least about 70%, 80%, 85%, 90%, or 95% sequenceidentity relative to the sequences identified in the cited references,or which exhibit substantially the same biological properties orcharacteristics as a TAA having a sequence found in the citedreferences. For example, a TAA having a variant sequence generally isable to bind specifically to an antibody that binds specifically to theTAA with the corresponding sequence listed. The sequences and disclosurein the reference specifically recited herein are expressly incorporatedby reference.

Tumor-Associated Antigens:

-   (1) BMPR1B (bone morphogenetic protein receptor-type IB, Genbank    accession no. NM_001203) ten Dijke, P., et al Science 264    (5155):101-104 (1994), Oncogene 14 (11):1377-1382 (1997));    WO2004063362 (claim 2); WO2003042661 (claim 12); US2003134790-A1    (Page 38-39); WO2002102235 (claim 13; Page 296); WO2003055443 (Page    91-92); WO200299122 (Example 2; Page 528-530); WO2003029421 (claim    6); WO2003024392 (claim 2; FIG. 112); WO200298358 (claim 1; Page    183); WO200254940 (Page 100-101); WO200259377 (Page 349-350);    WO200230268 (claim 27; Page 376); WO200148204 (Example; FIG. 4)    NP_001194 bone morphogenetic protein receptor, type    IB/pid=NP_001194.1—Cross-references: MIM:603248; NP_001194.1;    AY065994-   (2) E16 (LAT1, SLC7A5, Genbank accession no. NM_003486) Biochem.    Biophys. Res. Commun. 255 (2), 283-288 (1999), Nature 395    (6699):288-291 (1998), Gaugitsch, H. W., et al (1992) J. Biol. Chem.    267 (16):11267-11273); WO2004048938 (Example 2); WO2004032842    (Example IV); WO2003042661 (claim 12); WO2003016475 (claim 1);    WO200278524 (Example 2); WO200299074 (claim 19; Page 127-129);    WO200286443 (claim 27; Pages 222, 393); WO2003003906 (claim 10; Page    293); WO200264798 (claim 33; Page 93-95); WO200014228 (claim 5; Page    133-136); US2003224454 (FIG. 3); WO2003025138 (claim 12; Page 150);    NP_003477 solute carrier family 7 (cationic amino acid transporter,    y+ system), member 5/pid=NP_003477.3 —Homo sapiens Cross-references:    MIM:600182; NP_003477.3; NM_015923; NM_003486_1-   (3) STEAP1 (six transmembrane epithelial antigen of prostate,    Genbank accession no. NM_012449) Cancer Res. 61 (15), 5857-5860    (2001), Hubert, R. S., et al (1999) Proc. Natl. Acad. Sci. U.S.A. 96    (25):14523-14528); WO2004065577 (claim 6); WO2004027049 (FIG. 1L);    EP1394274 (Example 11); WO2004016225 (claim 2); WO2003042661 (claim    12); US2003157089 (Example 5); US2003185830 (Example 5);    US2003064397 (FIG. 2); WO200289747 (Example 5; Page 618-619);    WO2003022995 (Example 9; FIG. 13A, Example 53; Page 173, Example 2;    FIG. 2A); NP_036581 six transmembrane epithelial antigen of the    prostate Cross-references: MIM:604415; NP_036581.1; NM_012449_1-   (4) 0772P (CA125, MUC16, Genbank accession no. AF361486) J. Biol.    Chem. 276 (29):27371-27375 (2001)); WO2004045553 (claim 14);    WO200292836 (claim 6; FIG. 12); WO200283866 (claim 15; Page    116-121); US2003124140 (Example 16); US 798959. Cross-references:    GI:34501467; AAK74120.3; AF361486_1-   (5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor,    mesothelin, Genbank accession no. NM_005823) Yamaguchi, N., et al    Biol. Chem. 269 (2), 805-808 (1994), Proc. Natl. Acad. Sci. U.S.A.    96 (20):11531-11536 (1999), Proc. Natl. Acad. Sci. U.S.A. 93    (1):136-140 (1996), J. Biol. Chem. 270 (37):21984-21990 (1995));    WO2003101283 (claim 14); (WO2002102235 (claim 13; Page 287-288);    WO2002101075 (claim 4; Page 308-309); WO200271928 (Page 320-321);    WO9410312 (Page 52-57); Cross-references: MIM:601051; NP_005814.2;    NM_005823_1-   (6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34    (sodium phosphate), member 2, type II sodium-dependent phosphate    transporter 3b, Genbank accession no. NM_006424) J. Biol. Chem. 277    (22):19665-19672 (2002), Genomics 62 (2):281-284 (1999), Feild, J.    A., et al (1999) Biochem. Biophys. Res. Commun. 258 (3):578-582);    WO2004022778 (claim 2); EP1394274 (Example 11); WO2002102235 (claim    13; Page 326); EP875569 (claim 1; Page 17-19); WO200157188 (claim    20; Page 329); WO2004032842 (Example IV); WO200175177 (claim 24;    Page 139-140); Cross-references: MIM:604217; NP_006415.1;    NM_006424_1-   (7) Sema 5b (F1110372, KIAA1445, Mm.42015, SEMA5B, SEMAG, Semaphorin    5b Hlog, sema domain, seven thrombospondin repeats (type 1 and type    1-like), transmembrane domain (TM) and short cytoplasmic domain,    (semaphorin) 5B, Genbank accession no. AB040878) Nagase T., et    al (2000) DNA Res. 7 (2):143-150); WO2004000997 (claim 1);    WO2003003984 (claim 1); WO200206339 (claim 1; Page 50); WO200188133    (claim 1; Page 41-43, 48-58); WO2003054152 (claim 20); WO2003101400    (claim 11); Accession: Q9P283; EMBL; AB040878; BAA95969.1. Genew;    HGNC:10737;-   (8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12,    RIKEN cDNA 2700050C12 gene, Genbank accession no. AY358628); Ross et    al (2002) Cancer Res. 62:2546-2553; US2003129192 (claim 2);    US2004044180 (claim 12); US2004044179 (claim 11); US2003096961    (claim 11); US2003232056 (Example 5); WO2003105758 (claim 12);    US2003206918 (Example 5); EP1347046 (claim 1); WO2003025148 (claim    20); Cross-references: GI:37182378; AAQ88991.1; AY358628_1-   (9) ETBR (Endothelin type B receptor, Genbank accession no.    AY275463); Nakamuta M., et al Biochem. Biophys. Res. Commun. 177,    34-39, 1991; Ogawa Y., et al Biochem. Biophys. Res. Commun. 178,    248-255, 1991; Arai H., et al Jpn. Circ. J. 56, 1303-1307, 1992;    Arai H., et al J. Biol. Chem. 268, 3463-3470, 1993; Sakamoto A.,    Yanagisawa M., et al Biochem. Biophys. Res. Commun. 178, 656-663,    1991; Elshourbagy N. A., et al J. Biol. Chem. 268, 3873-3879, 1993;    Haendler B., et al J. Cardiovasc. Pharmacol. 20, s1-S4, 1992;    Tsutsumi M., et al Gene 228, 43-49, 1999; Strausberg R. L., et al    Proc. Natl. Acad. Sci. U.S.A. 99, 16899-16903, 2002; Bourgeois C.,    et al J. Clin. Endocrinol. Metab. 82, 3116-3123, 1997; Okamoto Y.,    et al Biol. Chem. 272, 21589-21596, 1997; Verheij J. B., et al    Am. J. Med. Genet. 108, 223-225, 2002; Hofstra R. M. W., et al    Eur. J. Hum. Genet. 5, 180-185, 1997; Puffenberger E. G., et al Cell    79, 1257-1266, 1994; Attie T., et al, Hum. Mol. Genet. 4, 2407-2409,    1995; Auricchio A., et al Hum. Mol. Genet. 5:351-354, 1996; Amiel    J., et al Hum. Mol. Genet. 5, 355-357, 1996; Hofstra R. M. W., et al    Nat. Genet. 12, 445-447, 1996; Svensson P. J., et al Hum. Genet.    103, 145-148, 1998; Fuchs S., et al Mol. Med. 7, 115-124, 2001;    Pingault V., et al (2002) Hum. Genet. 111, 198-206; WO2004045516    (claim 1); WO2004048938 (Example 2); WO2004040000 (claim 151);    WO2003087768 (claim 1); WO2003016475 (claim 1); WO2003016475 (claim    1); WO200261087 (FIG. 1); WO2003016494 (FIG. 6); WO2003025138 (claim    12; Page 144); WO200198351 (claim 1; Page 124-125); EP522868 (claim    8; FIG. 2); WO200177172 (claim 1; Page 297-299); US2003109676; U.S.    Pat. No. 6,518,404 (FIG. 3); U.S. Pat. No. 5,773,223 (Claim 1a; Col    31-34); WO2004001004;-   (10) MSG783 (RNF124, hypothetical protein F1120315, Genbank    accession no. NM_017763); WO2003104275 (claim 1); WO2004046342    (Example 2); WO2003042661 (claim 12); WO2003083074 (claim 14; Page    61); WO2003018621 (claim 1); WO2003024392 (claim 2; FIG. 93);    WO200166689 (Example 6); Cross-references: LocusID:54894;    NP_060233.2; NM_017763_1-   (11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP,    prostate cancer associated gene 1, prostate cancer associated    protein 1, six transmembrane epithelial antigen of prostate 2, six    transmembrane prostate protein, Genbank accession no. AF455138) Lab.    Invest. 82 (11):1573-1582 (2002)); WO2003087306; US2003064397 (claim    1; FIG. 1); WO200272596 (claim 13; Page 54-55); WO200172962 (claim    1; FIG. 4B); WO2003104270 (claim 11); WO2003104270 (claim 16);    US2004005598 (claim 22); WO2003042661 (claim 12); US2003060612    (claim 12; FIG. 10); WO200226822 (claim 23; FIG. 2); WO200216429    (claim 12; FIG. 10); Cross-references: GI:22655488; AAN04080.1;    AF455138_1-   (12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor    potential cation channel, subfamily M, member 4, Genbank accession    no. NM_017636) Xu, X. Z., et al Proc. Natl. Acad. Sci. U.S.A. 98    (19):10692-10697 (2001), Cell 109 (3):397-407 (2002), J. Biol. Chem.    278 (33):30813-30820 (2003)); US2003143557 (claim 4); WO200040614    (claim 14; Page 100-103); WO200210382 (claim 1; FIG. 9A);    WO2003042661 (claim 12); WO200230268 (claim 27; Page 391);    US2003219806 (claim 4); WO200162794 (claim 14; FIG. 1A-D);    Cross-references: MIM:606936; NP_060106.2; NM_017636_1-   (13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derived    growth factor, Genbank accession no. NP_003203 or NM_003212)    Ciccodicola, A., et al EMBO J. 8 (7):1987-1991 (1989), Am. J. Hum.    Genet. 49 (3):555-565 (1991)); US2003224411 (claim 1); WO2003083041    (Example 1); WO2003034984 (claim 12); WO200288170 (claim 2; Page    52-53); WO2003024392 (claim 2; FIG. 58); WO200216413 (claim 1; Page    94-95, 105); WO200222808 (claim 2; FIG. 1); U.S. Pat. No. 5,854,399    (Example 2; Col 17-18); U.S. Pat. No. 5,792,616 (FIG. 2);    Cross-references: MIM:187395; NP_003203.1; NM_003212_1-   (14) CD21 (CR2 (Complement receptor 2) or C3DR (C3d/Epstein Barr    virus receptor) or Hs.73792 Genbank accession no. M26004) Fujisaku    et al (1989) J. Biol. Chem. 264 (4):2118-2125); Weis J. J., et al J.    Exp. Med. 167, 1047-1066, 1988; Moore M., et al Proc. Natl. Acad.    Sci. U.S.A. 84, 9194-9198, 1987; Barel M., et al Mol. Immunol. 35,    1025-1031, 1998; Weis J. J., et al Proc. Natl. Acad. Sci. U.S.A. 83,    5639-5643, 1986; Sinha S. K., et al (1993) J. Immunol. 150,    5311-5320; WO2004045520 (Example 4); US2004005538 (Example 1);    WO2003062401 (claim 9); WO2004045520 (Example 4); WO9102536 (FIGS.    9.1-9.9); WO2004020595 (claim 1); Accession: P20023; Q13866; Q14212;    EMBL; M26004; AAA35786.1.-   (15) CD79b (CD79B, CD7913, IGb (immunoglobulin-associated beta),    B29, Genbank accession no. NM_000626 or 11038674) Proc. Natl. Acad.    Sci. U.S.A. (2003) 100 (7):4126-4131, Blood (2002) 100    (9):3068-3076, Muller et al (1992) Eur. J. Immunol. 22    (6):1621-1625); WO2004016225 (claim 2, FIG. 140); WO2003087768,    US2004101874 (claim 1, page 102); WO2003062401 (claim 9);    WO200278524 (Example 2); US2002150573 (claim 5, page 15); U.S. Pat.    No. 5,644,033; WO2003048202 (claim 1, pages 306 and 309); WO    99/558658, U.S. Pat. No. 6,534,482 (claim 13, FIG. 17A/B);    WO200055351 (claim 11, pages 1145-1146); Cross-references:    MIM:147245; NP_000617.1; NM_000626_1-   (16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing phosphatase    anchor protein 1a), SPAP1B, SPAP1C, Genbank accession no. NM_030764,    AY358130) Genome Res. 13 (10):2265-2270 (2003), Immunogenetics 54    (2):87-95 (2002), Blood 99 (8):2662-2669 (2002), Proc. Natl. Acad.    Sci. U.S.A. 98 (17):9772-9777 (2001), Xu, M. J., et al (2001)    Biochem. Biophys. Res. Commun. 280 (3):768-775; WO2004016225 (claim    2); WO2003077836; WO200138490 (claim 5; FIG. 18D-1-18D-2);    WO2003097803 (claim 12); WO2003089624 (claim 25); Cross-references:    MIM:606509; NP_110391.2; NM_030764_1-   (17) HER2 (ErbB2, Genbank accession no. M11730) Coussens L., et al    Science (1985) 230(4730):1132-1139); Yamamoto T., et al Nature 319,    230-234, 1986; Semba K., et al Proc. Natl. Acad. Sci. U.S.A. 82,    6497-6501, 1985; Swiercz J. M., et al J. Cell Biol. 165, 869-880,    2004; Kuhns J. J., et al J. Biol. Chem. 274, 36422-36427, 1999; Cho    H.-S., et al Nature 421, 756-760, 2003; Ehsani A., et al (1993)    Genomics 15, 426-429; WO2004048938 (Example 2); WO2004027049 (FIG.    1I); WO2004009622; WO2003081210; WO2003089904 (claim 9);    WO2003016475 (claim 1); US2003118592; WO2003008537 (claim 1);    WO2003055439 (claim 29; FIG. 1A-B); WO2003025228 (claim 37; FIG.    5C); WO200222636 (Example 13; Page 95-107); WO200212341 (claim 68;    FIG. 7); WO200213847 (Page 71-74); WO200214503 (Page 114-117);    WO200153463 (claim 2; Page 41-46); WO200141787 (Page 15);    WO200044899 (claim 52; FIG. 7); WO200020579 (claim 3; FIG. 2); U.S.    Pat. No. 5,869,445 (claim 3; Col 31-38); WO9630514 (claim 2; Page    56-61); EP1439393 (claim 7); WO2004043361 (claim 7); WO2004022709;    WO200100244 (Example 3; FIG. 4); Accession: P04626; EMBL; M11767;    AAA35808.1. EMBL; M11761; AAA35808.1.-   (18) NCA (CEACAM6, Genbank accession no. M18728); Barnett T., et al    Genomics 3, 59-66, 1988; Tawaragi Y., et al Biochem. Biophys. Res.    Commun. 150, 89-96, 1988; Strausberg R. L., et al Proc. Natl. Acad.    Sci. U.S.A. 99:16899-16903, 2002; WO2004063709; EP1439393 (claim 7);    WO2004044178 (Example 4); WO2004031238; WO2003042661 (claim 12);    WO200278524 (Example 2); WO200286443 (claim 27; Page 427);    WO200260317 (claim 2); Accession: P40199; Q14920; EMBL; M29541;    AAA59915.1. EMBL; M18728;-   (19) MDP (DPEP1, Genbank accession no. BC017023) Proc. Natl. Acad.    Sci. U.S.A. 99 (26):16899-16903 (2002)); WO2003016475 (claim 1);    WO200264798 (claim 33; Page 85-87); JP05003790 (FIG. 6-8); WO9946284    (FIG. 9); Cross-references: MIM:179780; AAH17023.1; BC017023_1-   (20) IL20Rα (IL20Ra, ZCYTOR7, Genbank accession no. AF184971);    Clark H. F., et al Genome Res. 13, 2265-2270, 2003; Mungall A. J.,    et al Nature 425, 805-811, 2003; Blumberg H., et al Cell 104, 9-19,    2001; Dumoutier L., et al J. Immunol. 167, 3545-3549, 2001;    Parrish-Novak J., et al J. Biol. Chem. 277, 47517-47523, 2002;    Pletnev S., et al (2003) Biochemistry 42:12617-12624; Sheikh F., et    al (2004) J. Immunol. 172, 2006-2010; EP1394274 (Example 11);    US2004005320 (Example 5); WO2003029262 (Page 74-75); WO2003002717    (claim 2; Page 63); WO200222153 (Page 45-47); US2002042366 (Page    20-21); WO200146261 (Page 57-59); WO200146232 (Page 63-65);    WO9837193 (claim 1; Page 55-59); Accession: Q9UHF4; Q6UWA9; Q96SH8;    EMBL; AF184971; AAF01320.1.-   (21) Brevican (BCAN, BEHAB, Genbank accession no. AF229053) Gary S.    C., et al Gene 256, 139-147, 2000; Clark H. F., et al Genome Res.    13, 2265-2270, 2003; Strausberg R. L., et al Proc. Natl. Acad. Sci.    U.S.A. 99, 16899-16903, 2002; US2003186372 (claim 11); US2003186373    (claim 11); US2003119131 (claim 1; FIG. 52); US2003119122 (claim 1;    FIG. 52); US2003119126 (claim 1); US2003119121 (claim 1; FIG. 52);    US2003119129 (claim 1); US2003119130 (claim 1); US2003119128 (claim    1; FIG. 52); US2003119125 (claim 1); WO2003016475 (claim 1);    WO200202634 (claim 1);-   (22) EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5, Genbank accession no.    NM_004442) Chan, J. and Watt, V. M., Oncogene 6 (6),    1057-1061 (1991) Oncogene 10 (5):897-905 (1995), Annu. Rev.    Neurosci. 21:309-345 (1998), Int. Rev. Cytol. 196:177-244 (2000));    WO2003042661 (claim 12); WO200053216 (claim 1; Page 41);    WO2004065576 (claim 1); WO2004020583 (claim 9); WO2003004529 (Page    128-132); WO200053216 (claim 1; Page 42); Cross-references:    MIM:600997; NP_004433.2; NM_004442_1-   (23) ASLG659 (B7h, Genbank accession no. AX092328) US20040101899    (claim 2); WO2003104399 (claim 11); WO2004000221 (FIG. 3);    US2003165504 (claim 1); US2003124140 (Example 2); US2003065143 (FIG.    60); WO2002102235 (claim 13; Page 299); US2003091580 (Example 2);    WO200210187 (claim 6; FIG. 10); WO200194641 (claim 12; FIG. 7b);    WO200202624 (claim 13; FIG. 1A-1B); US2002034749 (claim 54; Page    45-46); WO200206317 (Example 2; Page 320-321, claim 34; Page    321-322); WO200271928 (Page 468-469); WO200202587 (Example 1; FIG.    1); WO200140269 (Example 3; Pages 190-192); WO200036107 (Example 2;    Page 205-207); WO2004053079 (claim 12); WO2003004989 (claim 1);    WO200271928 (Page 233-234, 452-453); WO 0116318;-   (24) PSCA (Prostate stem cell antigen precursor, Genbank accession    no. AJ297436) Reiter R. E., et al Proc. Natl. Acad. Sci. U.S.A. 95,    1735-1740, 1998; Gu Z., et al Oncogene 19, 1288-1296, 2000; Biochem.    Biophys. Res. Commun. (2000) 275(3):783-788; WO2004022709; EP1394274    (Example 11); US2004018553 (claim 17); WO2003008537 (claim 1);    WO200281646 (claim 1; Page 164); WO2003003906 (claim 10; Page 288);    WO200140309 (Example 1; FIG. 17); US2001055751 (Example 1; FIG. 1b);    WO200032752 (claim 18; FIG. 1); WO9851805 (claim 17; Page 97);    WO9851824 (claim 10; Page 94); WO9840403 (claim 2; FIG. 1B);    Accession: 043653; EMBL; AF043498; AAC39607.1.-   (25) GEDA (Genbank accession No. AY260763); AAP14954 lipoma HMGIC    fusion-partner-like protein/pid=AAP14954.1 —Homo sapiens Species:    Homo sapiens (human) WO2003054152 (claim 20); WO2003000842 (claim    1); WO2003023013 (Example 3, claim 20); US2003194704 (claim 45);    Cross-references: GI:30102449; AAP14954.1; AY260763_1-   (26) BAFF-R (B cell-activating factor receptor, BLyS receptor 3,    BR3, Genbank accession No. AF116456); BAFF receptor/pid=NP_443177.1    —Homo sapiens Thompson, J. S., et al Science 293 (5537), 2108-2111    (2001); WO2004058309; WO2004011611; WO2003045422 (Example; Page    32-33); WO2003014294 (claim 35; FIG. 6B); WO2003035846 (claim 70;    Page 615-616); WO200294852 (Col 136-137); WO200238766 (claim 3; Page    133); WO200224909 (Example 3; FIG. 3); Cross-references: MIM:606269;    NP_443177.1; NM_052945_1; AF132600-   (27) CD22 (B-cell receptor CD22-B isoform, BL-CAM, Lyb-8, Lyb8,    SIGLEC-2, FLJ22814, Genbank accession No. AK026467); Wilson et    al (1991) J. Exp. Med. 173:137-146; WO2003072036 (claim 1; FIG. 1);    Cross-references: MIM:107266; NP_001762.1; NM_001771_1-   (28) CD79a (CD79A, CD79α, immunoglobulin-associated alpha, a B    cell-specific protein that covalently interacts with Ig beta (CD79B)    and forms a complex on the surface with Ig M molecules, transduces a    signal involved in B-cell differentiation), pI: 4.84, MW: 25028 TM:    2 [P] Gene Chromosome: 19q13.2, Genbank accession No. NP_001774.10)    WO2003088808, US20030228319; WO2003062401 (claim 9); US2002150573    (claim 4, pages 13-14); WO9958658 (claim 13, FIG. 16); WO9207574    (FIG. 1); U.S. Pat. No. 5,644,033; Ha et al (1992) J. Immunol.    148(5):1526-1531; Mueller et al (1992) Eur. J. Biochem.    22:1621-1625; Hashimoto et al (1994) Immunogenetics 40(4):287-295;    Preud'homme et al (1992) Clin. Exp. Immunol. 90(1):141-146; Yu et    al (1992) J. Immunol. 148(2) 633-637; Sakaguchi et al (1988) EMBO J.    7(11):3457-3464;-   (29) CXCR5 (Burkitt's lymphoma receptor 1, a G protein-coupled    receptor that is activated by the CXCL13 chemokine, functions in    lymphocyte migration and humoral defense, plays a role in HIV-2    infection and perhaps development of AIDS, lymphoma, myeloma, and    leukemia); 372 aa, pI: 8.54 MW: 41959 TM: 7 [P] Gene Chromosome:    11q23.3, Genbank accession No. NP_001707.1) WO2004040000;    WO2004015426; US2003105292 (Example 2); U.S. Pat. No. 6,555,339    (Example 2); WO200261087 (FIG. 1); WO200157188 (claim 20, page 269);    WO200172830 (pages 12-13); WO200022129 (Example 1, pages 152-153,    Example 2, pages 254-256); WO9928468 (claim 1, page 38); U.S. Pat.    No. 5,440,021 (Example 2, col 49-52); WO9428931 (pages 56-58);    WO9217497 (claim 7, FIG. 5); Dobner et al (1992) Eur. J. Immunol.    22:2795-2799; Barella et al (1995) Biochem. J. 309:773-779;-   (30) HLA-DOB (Beta subunit of MHC class II molecule (Ia antigen)    that binds peptides and presents them to CD4+ T lymphocytes); 273    aa, pI: 6.56 MW: 30820 TM: 1 [P] Gene Chromosome: 6p21.3, Genbank    accession No. NP_002111.1) Tonnelle et al (1985) EMBO J.    4(11):2839-2847; Jonsson et al (1989) Immunogenetics 29(6):411-413;    Beck et al (1992) J. Mol. Biol. 228:433-441; Strausberg et al (2002)    Proc. Natl. Acad. Sci USA 99:16899-16903; Servenius et al (1987) J.    Biol. Chem. 262:8759-8766; Beck et al (1996) J. Mol. Biol. 255:1-13;    Naruse et al (2002) Tissue Antigens 59:512-519; WO9958658 (claim 13,    FIG. 15); U.S. Pat. No. 6,153,408 (Col 35-38); U.S. Pat. No.    5,976,551 (col 168-170); U.S. Pat. No. 6,011,146 (col 145-146);    Kasahara et al (1989) Immunogenetics 30(1):66-68; Larhammar et    al (1985) J. Biol. Chem. 260(26):14111-14119;-   (31) P2X5 (Purinergic receptor P2X ligand-gated ion channel 5, an    ion channel gated by extracellular ATP, may be involved in synaptic    transmission and neurogenesis, deficiency may contribute to the    pathophysiology of idiopathic detrusor instability); 422 aa), pI:    7.63, MW: 47206 TM: 1 [P] Gene Chromosome: 17p13.3, Genbank    accession No. NP_002552.2) Le et al (1997) FEBS Lett.    418(1-2):195-199; WO2004047749; WO2003072035 (claim 10); Touchman et    al (2000) Genome Res. 10:165-173; WO200222660 (claim 20);    WO2003093444 (claim 1); WO2003087768 (claim 1); WO2003029277 (page    82);-   (32) CD72 (B-cell differentiation antigen CD72, Lyb-2) PROTEIN    SEQUENCE Full maeaity (SEQ ID NO: 29) . . . tafrfpd (SEQ ID NO: 30)    (1 . . . 359; 359 aa), p1: 8.66, MW: 40225 TM: 1 [P] Gene    Chromosome: 9p13.3, Genbank accession No. NP_001773.1) WO2004042346    (claim 65); WO2003026493 (pages 51-52, 57-58); WO200075655 (pages    105-106); Von Hoegen et al (1990) J. Immunol. 144(12):4870-4877;    Strausberg et al (2002) Proc. Natl. Acad. Sci USA 99:16899-16903;-   (33) LY64 (Lymphocyte antigen 64 (RP105), type I membrane protein of    the leucine rich repeat (LRR) family, regulates B-cell activation    and apoptosis, loss of function is associated with increased disease    activity in patients with systemic lupus erythematosis); 661 aa, p1:    6.20, MW: 74147 TM: 1 [P] Gene Chromosome: 5q12, Genbank accession    No. NP_005573.1) US2002193567; WO9707198 (claim 11, pages 39-42);    Miura et al (1996) Genomics 38(3):299-304; Miura et al (1998) Blood    92:2815-2822; WO2003083047; WO9744452 (claim 8, pages 57-61);    WO200012130 (pages 24-26);-   (34) FcRH1 (Fc receptor-like protein 1, a putative receptor for the    immunoglobulin Fc domain that contains C2 type Ig-like and ITAM    domains, may have a role in B-lymphocyte differentiation); 429 aa,    p1: 5.28, MW: 46925 TM: 1 [P] Gene Chromosome: 1q21-1q22, Genbank    accession No. NP_443170.1) WO2003077836; WO200138490 (claim 6, FIG.    18E-1-18-E-2); Davis et al (2001) Proc. Natl. Acad. Sci USA    98(17):9772-9777; WO2003089624 (claim 8); EP1347046 (claim 1);    WO2003089624 (claim 7);-   (35) IRTA2 (Immunoglobulin superfamily receptor translocation    associated 2, a putative immunoreceptor with possible roles in B    cell development and lymphomagenesis; deregulation of the gene by    translocation occurs in some B cell malignancies); 977 aa, p1: 6.88    MW: 106468 TM: 1 [P] Gene Chromosome: 1q21, Genbank accession No.    Human:AF343662, AF343663, AF343664, AF343665, AF369794, AF397453,    AK090423, AK090475, AL834187, AY358085; Mouse:AK089756, AY158090,    AY506558; NP_112571.1 WO2003024392 (claim 2, FIG. 97); Nakayama et    al (2000) Biochem. Biophys. Res. Commun. 277(1):124-127;    WO2003077836; WO200138490 (claim 3, FIG. 18B-1-18B-2);-   (36) TENB2 (TMEFF2, tomoregulin, TPEF, HPP1, TR, putative    transmembrane proteoglycan, related to the EGF/heregulin family of    growth factors and follistatin); 374 aa, NCBI Accession: AAD55776,    AAF91397, AAG49451, NCBI RefSeq: NP_057276; NCBI Gene: 23671; OMIM:    605734; SwissProt Q9UIK5; Genbank accession No. AF179274; AY358907,    CAF85723, CQ782436 WO2004074320 (SEQ ID NO 810); JP2004113151 (SEQ    ID NOS 2, 4, 8); WO2003042661 (SEQ ID NO 580); WO2003009814 (SEQ ID    NO 411); EP1295944 (pages 69-70); WO200230268 (page 329);    WO200190304 (SEQ ID NO 2706); US2004249130; US2004022727;    WO2004063355; US2004197325; US2003232350; US2004005563;    US2003124579; Horie et al (2000) Genomics 67:146-152; Uchida et    al (1999) Biochem. Biophys. Res. Commun. 266:593-602; Liang et    al (2000) Cancer Res. 60:4907-12; Glynne-Jones et al (2001) Int J    Cancer. October 15; 94(2):178-84;-   (37) PMEL17 (silver homolog; SILV; D12S53E; PMEL17; SI; SIL); ME20;    gp100) BC001414; BT007202; M32295; M77348; NM_006928;    McGlinchey, R. P. et al (2009) Proc. Natl. Acad. Sci. U.S.A. 106    (33), 13731-13736; Kummer, M. P. et al (2009) J. Biol. Chem. 284    (4), 2296-2306;-   (38) TMEFF1 (transmembrane protein with EGF-like and two    follistatin-like domains 1; Tomoregulin-1); H7365; C9orf2; C9ORF2;    U19878; X83961; NM_080655; NM_003692; Harms, P. W. (2003) Genes Dev.    17 (21), 2624-2629; Gery, S. et al (2003) Oncogene 22    (18):2723-2727;-   (39) GDNF-Ra1 (GDNF family receptor alpha1; GFRA1; GDNFR; GDNFRA;    RETL1; TRNR1; RET1L; GDNFR-alpha1; GFR-ALPHA-1); U95847; BC014962;    NM_145793 NM_005264; Kim, M. H. et al (2009) Mol. Cell. Biol. 29    (8), 2264-2277; Treanor, J. J. et al (1996) Nature 382 (6586):80-83;-   (40) Ly6E (lymphocyte antigen 6 complex, locus E; Ly67, RIG-E,    SCA-2, TSA-1); NP_002337.1; NM_002346.2; de Nooij-van Dalen, A. G.    et al (2003) Int. J. Cancer 103 (6), 768-774; Zammit, D. J. et    al (2002) Mol. Cell. Biol. 22 (3):946-952;-   (41) TMEM46 (shisa homolog 2 (Xenopus laevis); SHISA2);    NP_001007539.1; NM_001007538.1; Furushima, K. et al (2007) Dev.    Biol. 306 (2), 480-492; Clark, H. F. et al (2003) Genome Res. 13    (10):2265-2270;-   (42) Ly6G6D (lymphocyte antigen 6 complex, locus G6D; Ly6-D, MEGT1);    NP_067079.2; NM_021246.2; Mallya, M. et al (2002) Genomics 80    (1):113-123; Ribas, G. et al (1999) J. Immunol. 163 (1):278-287;-   (43) LGR5 (leucine-rich repeat-containing G protein-coupled receptor    5; GPR49, GPR67); NP_003658.1; NM_003667.2; Salanti, G. et al (2009)    Am. J. Epidemiol. 170 (5):537-545; Yamamoto, Y. et al (2003)    Hepatology 37 (3):528-533;-   (44) RET (ret proto-oncogene; MEN2A; HSCR1; MEN2B; MTC1; PTC;    CDHF12; Hs.168114; RET51; RET-ELE1); NP_066124.1; NM_020975.4;    Tsukamoto, H. et al (2009) Cancer Sci. 100 (10):1895-1901;    Narita, N. et al (2009) Oncogene 28 (34):3058-3068;-   (45) LY6K (lymphocyte antigen 6 complex, locus K; LY6K; HSJ001348;    FLJ35226); NP_059997.3; NM_017527.3; Ishikawa, N. et al (2007)    Cancer Res. 67 (24):11601-11611; de Nooij-van Dalen, A. G. et    al (2003) Int. J. Cancer 103 (6):768-774;-   (46) GPR19 (G protein-coupled receptor 19; Mm.4787); NP_006134.1;    NM_006143.2; Montpetit, A. and Sinnett, D. (1999) Hum. Genet. 105    (1-2):162-164; O'Dowd, B. F. et al (1996) FEBS Lett. 394    (3):325-329;-   (47) GPR54 (KiSS1 receptor; KISS1R; GPR54; HOT7T175; AXOR12);    NP_115940.2; NM_032551.4; Navenot, J. M. et al (2009) Mol.    Pharmacol. 75 (6):1300-1306; Hata, K. et al (2009) Anticancer Res.    29 (2):617-623;-   (48) ASPHD1 (aspartate beta-hydroxylase domain containing;    LOC253982); NP_859069.2; NM_181718.3; Gerhard, D. S. et al (2004)    Genome Res. 14 (10B):2121-2127;-   (49) Tyrosinase (TYR; OCAIA; OCA1A; tyrosinase; SHEP3); NP_000363.1;    NM_000372.4; Bishop, D. T. et al (2009) Nat. Genet. 41 (8):920-925;    Nan, H. et al (2009) Int. J. Cancer 125 (4):909-917;-   (50) TMEM118 (ring finger protein, transmembrane 2; RNFT2;    FLJ14627); NP_001103373.1; NM_001109903.1; Clark, H. F. et al (2003)    Genome Res. 13 (10):2265-2270; Scherer, S. E. et al (2006) Nature    440 (7082):346-351-   (51) GPR172A (G protein-coupled receptor 172A; GPCR41; FLJ11856;    D15Ertd747e); NP_078807.1; NM_024531.3; Ericsson, T. A. et al (2003)    Proc. Natl. Acad. Sci. U.S.A. 100 (11):6759-6764; Takeda, S. et    al (2002) FEBS Lett. 520 (1-3):97-101.-   (52) CD33, a member of the sialic acid binding, immunoglobulin-like    lectin family, is a 67-kDa glycosylated transmembrane protein. CD33    is expressed on most myeloid and monocytic leukemia cells in    addition to committed myelomonocytic and erythroid progenitor cells.    It is not seen on the earliest pluripotent stem cells, mature    granulocytes, lymphoid cells, or nonhematopoietic cells (Sabbath et    al., (1985) J. Clin. Invest. 75:756-56; Andrews et al., (1986) Blood    68:1030-5). CD33 contains two tyrosine residues on its cytoplasmic    tail, each of which is followed by hydrophobic residues similar to    the immunoreceptor tyrosine-based inhibitory motif (ITIM) seen in    many inhibitory receptors.-   (53) CLL-1 (CLEC12A, MICL, and DCAL2), encodes a member of the    C-type lectin/C-type lectin-like domain (CTL/CTLD) superfamily.    Members of this family share a common protein fold and have diverse    functions, such as cell adhesion, cell-cell signalling, glycoprotein    turnover, and roles in inflammation and immune response. The protein    encoded by this gene is a negative regulator of granulocyte and    monocyte function. Several alternatively spliced transcript variants    of this gene have been described, but the full-length nature of some    of these variants has not been determined. This gene is closely    linked to other CTL/CTLD superfamily members in the natural killer    gene complex region on chromosome 12p13 (Drickamer K (1999) Curr.    Opin. Struct. Biol. 9 (5):585-90; van Rhenen A, et al., (2007) Blood    110 (7):2659-66; Chen C H, et al. (2006) Blood 107 (4):1459-67;    Marshall A S, et al. (2006) Eur. J. Immunol. 36 (8):2159-69; Bakker    A B, et al (2005) Cancer Res. 64 (22):8443-50; Marshall A S, et    al (2004) J. Biol. Chem. 279 (15):14792-802). CLL-1 has been shown    to be a type II transmembrane receptor comprising a single C-type    lectin-like domain (which is not predicted to bind either calcium or    sugar), a stalk region, a transmembrane domain and a short    cytoplasmic tail containing an ITIM motif.

Anti-CD22 Antibodies

The anti-CD22 antibodies of ADC in Table 3 comprise three light chainhypervariable regions (HVR-L1, HVR-L2 and HVR-L3) and three heavy chainhypervariable regions (HVR-H1, HVR-H2 and HVR-H3), according to U.S.Pat. No. 8,226,945:

HVR-L1 (SEQ ID NO: 1) RSSQSIVHSVGNTFLE HVR-L2 (SEQ ID NO: 2) KVSNRFSHVR-L3 (SEQ ID NO: 3) FQGSQFPYT HVR-H1 (SEQ ID NO: 4) GYEFSRSWMN HVR-H2(SEQ ID NO: 5) GRIYPGDGDTNYSGKFKG HVR-H3 (SEQ ID NO: 6) DGSSWDWYFDV

Anti-HER2 Antibodies

In certain embodiments, ADC of Table 3 comprise anti-HER2 antibodies. Inone embodiment of the invention, an anti-HER2 antibody of an ADC of theinvention comprises a humanized anti-HER2 antibody, e.g., huMAb4D5-1,huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7and huMAb4D5-8, as described in Table 3 of U.S. Pat. No. 5,821,337,which is specifically incorporated by reference herein. Those antibodiescontain human framework regions with the complementarity-determiningregions of a murine antibody (4D5) that binds to HER2. The humanizedantibody huMAb4D5-8 is also referred to as trastuzumab, commerciallyavailable under the tradename HERCEPTIN®. In another embodiment of theinvention, an anti-HER2 antibody of an ADC of the invention comprises ahumanized anti-HER2 antibody, e.g., humanized 2C4, as described in U.S.Pat. No. 7,862,817. An exemplary humanized 2C4 antibody is pertuzumab,commercially available under the tradename PERJETA®.

In another embodiment of the invention, an anti-HER2 antibody of an ADCof the invention comprises a humanized anti-HER2 antibody is 7C2.

In some embodiments, cysteine-engineered THIOMAB™ antibodies used toprepare the ADC of Table 3 have a cysteine residue introduced at the149-lysine site of the light chain (LC K149C). In other embodiments, thecysteine-engineered THIOMAB™ antibodies have a cysteine residueintroduced at the 118-alanine site (EU numbering) of the heavy chain (HCA118C). This site is alternatively numbered 121 by Sequential numberingor 114 by Kabat numbering.

Anti-CD33 Antibodies

The anti-CD33 antibody 15G15.33 of ADC in Table 2 comprises three lightchain hypervariable regions (HVR-L1, HVR-L2 and HVR-L3) and three heavychain hypervariable regions (HVR-H1, HVR-H2 and HVR-H3)

HVR-L1 (SEQ ID NO: 7) RSSQSLLHSNGYNYLD HVR-L2 (SEQ ID NO: 8) LGVNSVSHVR-L3 (SEQ ID NO: 9) MQALQTPWT HVR-H1 (SEQ ID NO: 10) NHAIS HVR-H2(SEQ ID NO: 11) GIIPIFGTANYAQKFQG HVR-H3 (SEQ ID NO: 12) EWADVFD

The anti-CD33 antibody 15G15.33 of ADC in Table 5 comprises the lightchain variable region of SEQ ID NO:13 and/or the heavy chain variableregion of SEQ ID NO:14.

(SEQ ID NO: 13) EIVLTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGVNSVSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQ TPWTFGQGTKVEIK(SEQ ID NO: 14) QVQLVQSGAEVKKPGSSVKVSCKASGGIFSNHAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAFMELSSLRSEDTAVYYCAR EWADVFDIWGQGTMVTVSS

Anti-CD33 Antibody 9C3 and Other Embodiments

9C3-HVR L1 (SEQ ID NO: 15) RASQGIRNDLG 9C3-HVR L2 (SEQ ID NO: 16)AASSLQS 9C3-HVR L3 (SEQ ID NO: 17) LQHNSYPWT 9C3-HVR H1 (SEQ ID NO: 18)GNYMS 9C3-HVR H2 (SEQ ID NO: 19) LIYSGDSTYYADSVKG 9C3-HVR H3(SEQ ID NO: 20) DGYYVSDMVV 9C3 V_(L) (SEQ ID NO: 21)DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPWTF GQGTKLEIK 9C3 V_(H)(SEQ ID NO: 22) EVQLVESGGALIQPGGSLRLSCVASGFTISGNYMSWVRQAPGKGLEWVSLIYSGDSTYYADSVKGRFNISRDISKNTVYLQMNSLRVEDTAVYYCVRD GYYVSDMVVWGKGTTVTVSS9C3.2 V_(L) (SEQ ID NO: 23)DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPWTF GQGTKLEIK 9C3.2 V_(H)(SEQ ID NO: 24) EVQLVESGGALIQPGGSLRLSCVASGFTISGNYMSWVRQAPGKGLEWVSLIYSGDSTYYADSVKGRFTISRDISKNTVYLQMNSLRVEDTAVYYCVRD GYYVSDMVVWGKGTTVTVSS9C3.3 V_(L) (SEQ ID NO: 25)DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPWTF GQGTKLEIK 9C3.3 V_(H)(SEQ ID NO: 26) EVQLVESGGALIQPGGSLRLSCVASGFTISGNYMSWVRQAPGKGLEWVSLIYSGDSTYYADSVKGRFSISRDISKNTVYLQMNSLRVEDTAVYYCVRD GYYVSDMVVWGKGTTVTVSS9C3.4 V_(L) (SEQ ID NO: 27)DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPWTF GQGTKLEIK 9C3.4 V_(H)(SEQ ID NO: 28) EVQLVESGGALIQPGGSLRLSCVASGFTISGNYMSWVRQAPGKGLEWVSLIYSGDSTYYADSVKGRFAISRDISKNTVYLQMNSLRVEDTAVYYCVRD GYYVSDMVVWGKGTTVTVSS

In some embodiments, the invention provides an anti-CD33 antibodycomprising at least one, two, three, four, five, or six HVRs selectedfrom (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:18; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO:19; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:20; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:15; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:16; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:17.

In one aspect, the invention provides an antibody comprising at leastone, at least two, or all three VH HVR sequences selected from (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:18; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:19; and (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:20. In one embodiment,the antibody comprises HVR-H3 comprising the amino acid sequence of SEQID NO:20. In another embodiment, the antibody comprises HVR-H3comprising the amino acid sequence of SEQ ID NO:20 and HVR-L3 comprisingthe amino acid sequence of SEQ ID NO:17. In a further embodiment, theantibody comprises HVR-H3 comprising the amino acid sequence of SEQ IDNO:20, HVR-L3 comprising the amino acid sequence of SEQ ID NO:17, andHVR-H2 comprising the amino acid sequence of SEQ ID NO:19. In a furtherembodiment, the antibody comprises (a) HVR-H1 comprising the amino acidsequence of SEQ ID NO:18; (b) HVR-H2 comprising the amino acid sequenceof SEQ ID NO:19; and (c) HVR-H3 comprising the amino acid sequence ofSEQ ID NO:20.

In another aspect, the invention provides an antibody comprising atleast one, at least two, or all three VL HVR sequences selected from (a)HVR-L1 comprising the amino acid sequence of SEQ ID NO:15; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO:16; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO:17. In one embodiment,the antibody comprises (a) HVR-L1 comprising the amino acid sequence ofSEQ ID NO:15; (b) HVR-L2 comprising the amino acid sequence of SEQ IDNO:16; and (c) HVR-L3 comprising the amino acid sequence of SEQ IDNO:17.

In another aspect, an antibody of the invention comprises (a) a VHdomain comprising at least one, at least two, or all three VH HVRsequences selected from (i) HVR-H1 comprising the amino acid sequence ofSEQ ID NO:18, (ii) HVR-H2 comprising the amino acid sequence of SEQ IDNO:19, and (iii) HVR-H3 comprising an amino acid sequence selected fromSEQ ID NO:20; and (b) a VL domain comprising at least one, at least two,or all three VL HVR sequences selected from (i) HVR-L1 comprising theamino acid sequence of SEQ ID NO:15, (ii) HVR-L2 comprising the aminoacid sequence of SEQ ID NO:16, and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:17.

In another aspect, the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:18; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:19; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:20; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:15; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:16; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:17.

In any of the above embodiments, an anti-CD33 antibody is humanized. Inone embodiment, an anti-CD33 antibody comprises HVRs as in any of theabove embodiments, and further comprises a human acceptor framework,e.g. a human immunoglobulin framework or a human consensus framework. Incertain embodiments, the human acceptor framework is the human VL kappaI consensus (VL_(KI)) framework and/or the VH framework VH₁. In certainembodiments, the human acceptor framework is the human VL kappa Iconsensus (VL_(KI)) framework and/or the VH framework VH₁ comprising anyone of the following mutations.

In another aspect, an anti-CD33 antibody comprises a heavy chainvariable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acidsequence of SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, and/or SEQ IDNO:28. In certain embodiments, a VH sequence having at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acidsequence of SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, and/or SEQ IDNO:28 contains substitutions (e.g., conservative substitutions),insertions, or deletions relative to the reference sequence, but ananti-CD33 antibody comprising that sequence retains the ability to bindto CD33. In certain embodiments, a total of 1 to 10 amino acids havebeen substituted, inserted and/or deleted in SEQ ID NO:22, SEQ ID NO:24,SEQ ID NO:26, and/or SEQ ID NO:28. In certain embodiments, a total of 1to 5 amino acids have been substituted, inserted and/or deleted in SEQID NO:22, SEQ ID NO:24, SEQ ID NO:26, and/or SEQ ID NO:28. In certainembodiments, substitutions, insertions, or deletions occur in regionsoutside the HVRs (i.e., in the FRs). Optionally, the anti-CD33 antibodycomprises the VH sequence of SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26,and/or SEQ ID NO:28, including post-translational modifications of thatsequence. In a particular embodiment, the VH comprises one, two or threeHVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQID NO:18, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:19,and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:20.

In another aspect, an anti-CD33 antibody is provided, wherein theantibody comprises a light chain variable domain (VL) having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO:21, SEQ ID NO:23, SEQID NO:25, and/or SEQ ID NO:27. In certain embodiments, a VL sequencehaving at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to the amino acid sequence of SEQ ID NO:21, SEQ ID NO:23, SEQID NO:25, and/or SEQ ID NO:27 contains substitutions (e.g., conservativesubstitutions), insertions, or deletions relative to the referencesequence, but an anti-CD33 antibody comprising that sequence retains theability to bind to CD33. In certain embodiments, a total of 1 to 10amino acids have been substituted, inserted and/or deleted in SEQ IDNO:21, SEQ ID NO:23, SEQ ID NO:25, and/or SEQ ID NO:27. In certainembodiments, a total of 1 to 5 amino acids have been substituted,inserted and/or deleted in SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25,and/or SEQ ID NO:27. In certain embodiments, the substitutions,insertions, or deletions occur in regions outside the HVRs (i.e., in theFRs). Optionally, the anti-CD33 antibody comprises the VL sequence ofSEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, and/or SEQ ID NO:27, includingpost-translational modifications of that sequence. In a particularembodiment, the VL comprises one, two or three HVRs selected from (a)HVR-L1 comprising the amino acid sequence of SEQ ID NO:15; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO:16; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO:17.

In another aspect, an anti-CD33 antibody is provided, wherein theantibody comprises a VH as in any of the embodiments provided above, anda VL as in any of the embodiments provided above.

In one embodiment, the antibody comprises the VH and VL sequences in SEQID NO:22 and SEQ ID NO:21, respectively, including post-translationalmodifications of those sequences. In one embodiment, the antibodycomprises the VH and VL sequences in SEQ ID NO:24 and SEQ ID NO:23,respectively, including post-translational modifications of thosesequences. In one embodiment, the antibody comprises the VH and VLsequences in SEQ ID NO:26 and SEQ ID NO:25, respectively, includingpost-translational modifications of those sequences. In one embodiment,the antibody comprises the VH and VL sequences in SEQ ID NO:28 and SEQID NO:27, respectively, including post-translational modifications ofthose sequences.

In a further aspect, provided are herein are antibodies that bind to thesame epitope as an anti-CD33 antibody provided herein. For example, incertain embodiments, an antibody is provided that binds to the sameepitope as an anti-CD33 antibody comprising a VH sequence of SEQ IDNO:22, SEQ ID NO:24, SEQ ID NO:26, and/or SEQ ID NO:28 and a VL sequenceof SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, and/or SEQ ID NO:27,respectively.

In a further aspect of the invention, an anti-CD33 antibody according toany of the above embodiments is a monoclonal antibody, including a humanantibody. In one embodiment, an anti-CD33 antibody is an antibodyfragment, e.g., a Fv, Fab, Fab′, scFv, diabody, or F(ab′)2 fragment. Inanother embodiment, the antibody is a substantially full lengthantibody, e.g., an IgG1 antibody, IgG2a antibody or other antibody classor isotype as defined herein.

In a further aspect, an anti-CD33 antibody according to any of the aboveembodiments may incorporate any of the features, singly or incombination, as described below.

The parent antibody may also be a fusion protein comprising analbumin-binding peptide (ABP) sequence (Dennis et al. (2002) J BiolChem. 277:35035-35043 (Tables III and IV, page 35038); and WO 2001/45746at pages 12-13, which are incorporated herein by reference.

To prepare a cysteine engineered antibody by mutagenesis, DNA encodingan amino acid sequence variant of the starting polypeptide is preparedby a variety of methods known in the art. These methods include, but arenot limited to, preparation by site-directed (oroligonucleotide-mediated) mutagenesis, PCR mutagenesis, and cassettemutagenesis of an earlier prepared DNA encoding the polypeptide.Variants of recombinant antibodies may be constructed also byrestriction fragment manipulation or by overlap extension PCR withsynthetic oligonucleotides. Mutagenic primers encode the cysteine codonreplacement(s). Standard mutagenesis techniques can be employed togenerate DNA encoding such mutant cysteine engineered antibodies.General guidance can be found in Sambrook et al Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989; and Ausubel et al Current Protocols in MolecularBiology, Greene Publishing and Wiley-Interscience, New York, N.Y., 1993.

Antibodies may be produced using recombinant methods and compositions,e.g., as described in U.S. Pat. No. 4,816,567. In one embodiment,isolated nucleic acid encodes an amino acid sequence comprising the VLand/or an amino acid sequence comprising the VH of the antibody (e.g.,the light and/or heavy chains of the antibody). In a further embodiment,one or more vectors (e.g., expression vectors) comprising such nucleicacid are provided. In a further embodiment, a host cell comprising suchnucleic acid is provided. In one such embodiment, a host cell comprises(e.g., has been transformed with): (1) a vector comprising a nucleicacid that encodes an amino acid sequence comprising the VL of theantibody and an amino acid sequence comprising the VH of the antibody,or (2) a first vector comprising a nucleic acid that encodes an aminoacid sequence comprising the VL of the antibody and a second vectorcomprising a nucleic acid that encodes an amino acid sequence comprisingthe VH of the antibody. In one embodiment, the host cell is eukaryotic,e.g. a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NS0,Sp20 cell). In one embodiment, a method comprises culturing a host cellcomprising a nucleic acid encoding the antibody, as provided above,under conditions suitable for expression of the antibody, and optionallyrecovering the antibody from the host cell (or host cell culturemedium).

For recombinant production, nucleic acid encoding an antibody, e.g., asdescribed above, is isolated and inserted into one or more vectors forfurther cloning and/or expression in a host cell. Such nucleic acid maybe readily isolated and sequenced using conventional procedures (e.g.,by using oligonucleotide probes that are capable of binding specificallyto genes encoding the heavy and light chains of the antibody). Suitablehost cells for cloning or expression of antibody-encoding vectorsinclude prokaryotic or eukaryotic cells described herein. For example,antibodies may be produced in bacteria, in particular when glycosylationand Fc effector function are not needed. For expression of antibodyfragments and polypeptides in bacteria, see, e.g., U.S. Pat. No.5,648,237, U.S. Pat. No. 5,789,199, and U.S. Pat. No. 5,840,523, alsoCharlton, Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed.,Humana Press, Totowa, N.J., 2003), pp. 245-254, describing expression ofantibody fragments in E. coli.) After expression, the antibody may beisolated from the bacterial cell paste in a soluble fraction and can befurther purified. In addition to prokaryotes, eukaryotic microbes suchas filamentous fungi or yeast are suitable cloning or expression hostsfor antibody-encoding vectors, including fungi and yeast strains whoseglycosylation pathways have been “humanized,” resulting in theproduction of an antibody with a partially or fully human glycosylationpattern. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li etal., Nat. Biotech. 24:210-215 (2006). Suitable host cells for theexpression of glycosylated antibody are also derived from multicellularorganisms (invertebrates and vertebrates). Examples of invertebratecells include plant and insect cells. Numerous baculoviral strains havebeen identified which may be used in conjunction with insect cells,particularly for transfection of Spodoptera frugiperda cells. Plant cellcultures can also be utilized as hosts, such as those described in U.S.Pat. No. 5,959,177, U.S. Pat. No. 6,040,498, U.S. Pat. No. 6,420,548,U.S. Pat. No. 7,125,978, and U.S. Pat. No. 6,417,429 (describingPLANTIBODIES™ technology for producing antibodies in transgenic plants).Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (293 or 293cells as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977);baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells asdescribed, e.g., in Mather, Biol. Reprod. 23:243-251 (1980); monkeykidney cells (CV1); African green monkey kidney cells (VERO-76); humancervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo ratliver cells (BRL 3A); human lung cells (W138); human liver cells (HepG2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., inMather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; andFS4 cells. Other useful mammalian host cell lines include Chinesehamster ovary (CHO) cells, including DHFR⁻ CHO cells (Urlaub et al.,Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines suchas Y0, NS0 and Sp2/0. For a review of certain mammalian host cell linessuitable for antibody production, see, e.g., Yazaki and Wu, Methods inMolecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa,N.J.), pp. 255-268 (2003).

Site-directed mutagenesis is one method for preparing substitutionvariants, i.e. mutant proteins (Carter (1985) et al Nucleic Acids Res.13:4431-4443; Ho et al (1989) Gene (Amst.) 77:51-59; and Kunkel et al(1987) Proc. Natl. Acad. Sci. USA 82:488). Starting DNA is altered byfirst hybridizing an oligonucleotide encoding the desired mutation to asingle strand of such starting DNA. After hybridization, a DNApolymerase is used to synthesize an entire second strand, using thehybridized oligonucleotide as a primer, and using the single strand ofthe starting DNA as a template. Thus, the oligonucleotide encoding thedesired mutation is incorporated in the resulting double-stranded DNA.Site-directed mutagenesis may be carried out within the gene expressingthe protein to be mutagenized in an expression plasmid and the resultingplasmid may be sequenced to confirm the introduction of the desiredcysteine replacement mutations (Liu et al (1998) J. Biol. Chem.273:20252-20260). Site-directed mutagenesis protocols and formats arewidely available, e.g. QuikChange® Multi Site-Directed Mutagenesis Kit(Stratagene, La Jolla, Calif.).

PCR mutagenesis is also suitable for making amino acid sequence variantsof the starting polypeptide. See Higuchi, (1990) in PCR Protocols, pp.177-183, Academic Press; Ito et al (1991) Gene 102:67-70; Bernhard et al(1994) Bioconjugate Chem., 5:126-132; and Vallette et al (1989) Nuc.Acids Res., 17:723-733. Briefly, when small amounts of template DNA areused as starting material in a PCR, primers that differ slightly insequence from the corresponding region in a template DNA can be used togenerate relatively large quantities of a specific DNA fragment thatdiffers from the template sequence only at the positions where theprimers differ from the template.

Another method for preparing variants, cassette mutagenesis, is based onthe technique described by Wells et al (1985) Gene, 34:315-323. Thestarting material is the plasmid (or other vector) comprising thestarting polypeptide DNA to be mutated. The codon(s) in the starting DNAto be mutated are identified. There must be a unique restrictionendonuclease site on each side of the identified mutation site(s). If nosuch restriction sites exist, they may be generated using the abovedescribed oligonucleotide-mediated mutagenesis method to introduce themat appropriate locations in the starting polypeptide DNA. The plasmidDNA is cut at these sites to linearize it. A double-strandedoligonucleotide encoding the sequence of the DNA between the restrictionsites but containing the desired mutation(s) is synthesized usingstandard procedures, wherein the two strands of the oligonucleotide aresynthesized separately and then hybridized together using standardtechniques. This double-stranded oligonucleotide is referred to as thecassette. This cassette is designed to have 5′ and 3′ ends that arecompatible with the ends of the linearized plasmid, such that it can bedirectly ligated to the plasmid. This plasmid now contains the mutatedDNA sequence. Mutant DNA containing the encoded cysteine replacementscan be confirmed by DNA sequencing.

Single mutations are also generated by oligonucleotide directedmutagenesis using double stranded plasmid DNA as template by PCR basedmutagenesis (Sambrook and Russel, (2001) Molecular Cloning: A LaboratoryManual, 3rd edition; Zoller et al (1983) Methods Enzymol. 100:468-500;Zoller, M. J. and Smith, M. (1982) Nucl. Acids Res. 10:6487-6500).

In certain embodiments, one or more amino acid modifications may beintroduced into the Fc region of an antibody provided herein, therebygenerating an Fc region variant. The Fc region variant may comprise ahuman Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fcregion) comprising an amino acid modification (e.g. a substitution) atone or more amino acid positions.

In certain embodiments, the invention contemplates an antibody variantthat possesses some but not all effector functions, which make it adesirable candidate for applications in which the half-life of theantibody in vivo is important yet certain effector functions (such ascomplement and ADCC) are unnecessary or deleterious. In vitro and/or invivo cytotoxicity assays can be conducted to confirm thereduction/depletion of CDC and/or ADCC activities. For example, Fcreceptor (FcR) binding assays can be conducted to ensure that theantibody lacks FcγR binding (hence likely lacking ADCC activity), butretains FcRn binding ability.

Anthracycline Disulfide Intermediates

An antibody-drug conjugate compound of the invention comprises ananthracycline disulfide intermediate comprised of a PNU-15682 moietyderivatized at the ketone group with a disulfide group.

The anthracycline disulfide intermediate has the formula:

Ant-L-(Z)_(m)—X  I

wherein Ant is selected from the structure:

where the wavy line indicates the attachment to L;

L is a linker selected from —CH₂O—, —CH₂N(R)—, —N(R)—, —N(R)(C₁-C₁₂alkylene)-, —N(R)(C₂-C₈ alkenylene)-, —N(R)(C₂-C₈ alkynylene)-,—N(R)(CH₂CH₂O)_(n)—, and the structure:

where the wavy lines indicate the attachments to Ant and Z; and

Z is an optional spacer selected from —CH₂C(O)—, —CH₂C(O)NR(C₁-C₁₂alkylene)-, and the structure:

where the wavy lines indicate the attachments to L and X;

R is H, C₁-C₁₂ alkyl, or C₆-C₂₀ aryl;

Z¹ is selected from —(C₁-C₁₂ alkylene)-, —(C₂-C₈ alkenylene)-, —(C₂-C₈alkynylene)-, —O(C₁-C₁₂ alkylene). —O(C₂-C₈ alkenylene)-, —O(C₂-C₈alkynylene)-, and (CH₂CH₂O)_(n)—;

m is 0 or 1;

n is 1 to 6;

X is pyridyl disulfide where pyridyl is optionally substituted with oneor more groups selected from NO₂, Cl, F, CN, and Br; and

alkylene, alkenylene, alkynylene, alkyl, and aryl are optionallysubstituted with one or more groups selected from F, Cl, Br, N(CH₃)₂,NO₂, and OCH₃.

An exemplary embodiment of X is:

where the wavy lines indicate the attachments to L or Z;

R¹ is NO₂, Cl, F, CN or Br, and q is 0, 1, or 2.

Without being limited to a particular mechanism or effect, the presenceof an electron-withdrawing group R¹; NO₂, Cl, F, or Br on the pyridylring of an anthracycline disulfide intermediate accelerates reactionwith a cysteine thiol of a cysteine-engineered antibody. Where thecysteine thiol has been introduced at a hindered or less-reactive siteon the antibody, such anthracycline disulfide intermediate give moreefficient conjugation relative to a corresponding unsubstituted pyridylanalog (R¹═H).

Exemplary linker-drug intermediates are Formulas Ia-c:

where R¹ is NO₂, Cl, F, CN or Br; and q is 0, 1, or 2.

An exemplary linker-drug intermediate include wherein R¹ is NO₂ and q is1.

An exemplary linker-drug intermediate is Formula Id:

Exemplary anthracycline disulfide, linker-drug intermediates useful forpreparing antibody-drug conjugates of the invention are included inTable 1. The synthesis of anthracycline disulfide, linker-drug (LD)intermediates are described in Example 1.

TABLE 1 Anthracycline disulfide, Linker-drug intermediates LD No.Structure LD-51

LD-52

LD-53

LD-54

LD-55

LD-56

LD-57

LD-58

LD-59

LD-60

LD-61

LD-62

LD-63

Antibody-Drug Conjugates (ADC)

The antibody-drug conjugate (ADC) compounds of the invention comprise anantibody specific for a tumor-associated antigen linked to a potentanthracycline drug moiety, and include those with therapeutic activity,effective against a number of hyperproliferative disorders, includingcancer. The biological activity of the drug moiety is modulated byconjugation to an antibody. The ADC of the invention selectively deliveran effective dose of the anthracycline drug, or toxin, to a tumor cellor site whereby greater selectivity, i.e. a lower efficacious dose, maybe achieved while increasing the therapeutic index (“therapeuticwindow”). In an exemplary embodiment, the ADC compounds include acysteine-engineered antibody conjugated, i.e. covalently attached by alinker, to the anthracycline drug moiety.

An antibody-drug conjugate compound of the invention comprises anantibody covalently attached by a disulfide, linker L and an optionalspacer Z to one or more anthracycline derivative drug moieties D, thecompound having Formula II:

Ab-S—S—(Z_(m)-L-D)_(p)  II

or a pharmaceutically acceptable salt thereof, wherein:

Ab is an antibody which binds to one or more tumor-associated antigensor cell-surface receptors selected from (1)-(53):

(1) BMPR1B (bone morphogenetic protein receptor-type IB);

(2) E16 (LAT1, SLC7A5);

(3) STEAP1 (six transmembrane epithelial antigen of prostate);

(4) MUC16 (0772P, CA125);

(5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin);

(6) Napi2b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodiumphosphate), member 2, type II sodium-dependent phosphate transporter3b);

(7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, Semaphorin 5bHlog, sema domain, seven thrombospondin repeats (type 1 and type1-like), transmembrane domain (TM) and short cytoplasmic domain,(semaphorin) 5B);

(8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12, RIKENcDNA 2700050C12 gene);

(9) ETBR (Endothelin type B receptor);

(10) MSG783 (RNF124, hypothetical protein FLJ20315);

(11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostatecancer associated gene 1, prostate cancer associated protein 1, sixtransmembrane epithelial antigen of prostate 2, six transmembraneprostate protein);

(12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptorpotential cation channel, subfamily M, member 4);

(13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derivedgrowth factor);

(14) CD21 (CR2 (Complement receptor 2) or C3DR (C3d/Epstein Barr virusreceptor) or Hs 73792);

(15) CD79b (CD79B, CD7913, IGb (immunoglobulin-associated beta), B29);

(16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing phosphataseanchor protein 1a), SPAP1B, SPAP1C);

(17) HER2;

(18) NCA;

(19) MDP;

(20) IL20Rα;

(21) Brevican;

(22) EphB2R;

(23) ASLG659;

(24) PSCA;

(25) GEDA;

(26) BAFF-R (B cell-activating factor receptor, BLyS receptor 3, BR3);

(27) CD22 (B-cell receptor CD22-B isoform);

(28) CD79a (CD79A, CD79a, immunoglobulin-associated alpha);

(29) CXCR5 (Burkitt's lymphoma receptor 1);

(30) HLA-DOB (Beta subunit of MHC class II molecule (Ia antigen));

(31) P2X5 (Purinergic receptor P2X ligand-gated ion channel 5);

(32) CD72 (B-cell differentiation antigen CD72, Lyb-2);

(33) LY64 (Lymphocyte antigen 64 (RP105), type I membrane protein of theleucine rich repeat (LRR) family);

(34) FcRH1 (Fc receptor-like protein 1);

(35) FcRH5 (IRTA2, Immunoglobulin superfamily receptor translocationassociated 2);

(36) TENB2 (putative transmembrane proteoglycan); and

(37) PMEL17 (silver homolog; SILV; D12S53E; PMEL17; SI; SIL);

(38) TMEFF1 (transmembrane protein with EGF-like and twofollistatin-like domains 1; Tomoregulin-1);

(39) GDNF-Ra1 (GDNF family receptor alpha1; GFRA1; GDNFR; GDNFRA; RETL1;TRNR1; RET1L; GDNFR-alpha1; GFR-ALPHA-1);

(40) Ly6E (lymphocyte antigen 6 complex, locus E; Ly67, RIG-E, SCA-2,TSA-1);

(41) TMEM46 (shisa homolog 2 (Xenopus laevis); SHISA2);

(42) Ly6G6D (Lymphocyte antigen 6 complex, locus G6D; Ly6-D, MEGT1);

(43) LGR5 (leucine-rich repeat-containing G protein-coupled receptor 5;GPR49, GPR67);

(44) RET (ret proto-oncogene; MEN2A; HSCR1; MEN2B; MTC1; PTC; CDHF12;Hs.168114; RET51; RET-ELE1);

(45) LY6K (lymphocyte antigen 6 complex, locus K; LY6K; HSJ001348;FLJ35226);

(46) GPR19 (G protein-coupled receptor 19; Mm.4787);

(47) GPR54 (KISS1 receptor; KISS1R; GPR54; HOT7T175; AXOR12);

(48) ASPHD1 (aspartate beta-hydroxylase domain containing 1; LOC253982);

(49) Tyrosinase (TYR; OCAIA; OCA1A; tyrosinase; SHEP3);

(50) TMEM118 (ring finger protein, transmembrane 2; RNFT2; FLJ14627);

(51) GPR172A (G protein-coupled receptor 172A; GPCR41; FLJ11856;D15Ertd747e);

(52) CD33; and

(53) CLL-1;

D is an anthracycline derivative selected from the structure:

where the wavy line indicates the attachment to L;

L is a linker selected from —CH₂O—, —CH₂N(R)—, —N(R)—, —N(R)(C₁-C₁₂alkylene)-, —N(R)(C₂-C₈ alkenylene)-, —N(R)(C₂-C₈ alkynylene)-,—N(R)(CH₂CH₂O)_(n)—, and the structure:

where the wavy lines indicate the attachments to D and Z; and

Z is an optional spacer selected from —CH₂C(O)—, —CH₂C(O)NR(C₁-C₁₂alkylene)-, and the structures:

R is H, C₁-C₁₂ alkyl, or C₆-C₂₀ aryl;

Z¹ is selected from —(C₁-C₁₂ alkylene)-, —(C₂-C₈ alkenylene)-, —(C₂-C₈alkynylene)-, and —(CH₂CH₂O)_(n)—,

m is 0 or 1;

n is 1 to 6; and

p is an integer from 1 to 8.

TABLE 2 Antibody-drug conjugates (ADC) linker-drug LD No. ADC No. ADCformula (Table 1) DAR* ADC-101 Thio Hu Anti-Her2 4D5 HC 51 1.8A118C-(LD-51) ADC-102 Thio Hu Anti-CD22 10F4v3 HC 51 1.8 A118C-(LD-51)ADC-103 Thio Hu Anti-Her2 4D5 HC 52 1.8 A118C-(LD-52) ADC-104 Thio HuAnti-CD22 10F4v3 HC 52 1.7 A118C-(LD-52) ADC-105 Thio Hu Anti-CD33GM15.33 HC 56 1.7 A118C-(LD-56) ADC-106 Thio Hu Anti-Napi3b 10H1.11.4B56 1.6 HC A118C-(LD-56) ADC-107 Thio Hu Anti-Her2 7C2 HC 51 1.6A118C-(LD-51) ADC-108 Thio Hu Anti-Her2 7C2 LC K149C- 51 1.7 (LD-51)ADC-109 Thio Hu anti-Her2 7C2 LC K149C- 57 1.9 (LD-57) ADC-110 Thio Huanti-CD33 15G15.3 LC 57 1.9 K149C-(LD-57) ADC-111 Thio Hu anti-Her2 4D5HC A118C- 56 1.2 (LD-56) ADC-112 Thio Hu Anti-Her2 4D5 HC 52 1.8A118C-(LD-52) ADC-113 Thio Hu Anti-Napi3b 10H1.11.4B 51 1.9 HCA118C-(LD-51) ADC-114 Thio Hu anti-Her2 7C2 HC A118C- 57 1.9 (LD-57)ADC-115 Thio Hu Anti-CD33 15G15.3 LC 59 1.6 K149C-(LD-59) ADC-116 ThioHu anti-Her2 7C2 LC K149C- 59 1.5 (LD-59) ADC-117 Thio Hu anti-CLL-16E7.N54A LC 59 1.7 K149C-(LD-59) ADC-118 Thio Hu anti-CLL-1 6E7.N54A LC57 2.0 K149C-(LD-57) ADC-119 Thio Hu Anti-Her2 4D5 HC 61 1.9A118C-(LD-61) ADC-120 Thio Hu anti-CD22 10F4v3 LC 57 1.9 K149C-(LD-57)ADC-121 Thio Hu Anti-Napi3b 10H1.11.4B 57 1.9 LC K149C-(LD-57) ADC-122Thio Hu anti-CD22 10F4v3 LC 59 1.7 K149C-(LD-59) ADC-123 Thio HuAnti-Napi3b 10H1.11.4B 59 1.7 LC K149C-(LD-59) ADC-124 Thio Hu anti-CD2210F4v3 LC 58 1.9 K149C-(LD-58) ADC-125 Thio Hu Anti-Napi3b 10H1.11.4B 581.9 LC K149C-(LD-58) ADC-126 Thio Hu anti-CD22 10F4v3 LC 60 1.7K149C-(LD-60) ADC-127 Thio Hu Anti-Napi3b 10H1.11.4B 60 1.6 LCK149C-(LD-60) ADC-128 Thio Hu-Anti 4D5 HC A140C-(LD- 59 1.2 59) ADC-129Thio Hu-Anti 4D5 LC K149C-(LD- 59 1.5 59) ADC-130 Thio Hu-Anti 4D5 LCV205C-(LD- 59 1.2 59) ADC-131 Thio Hu-Anti 4D5 HC A118C-(LD- 59 1.6 59)ADC-132 Thio Hu-Anti 4D5 LC S121C-(LD- 59 0.5 59) ADC-133 Thio Huanti-CLL-1 6E7.N54A LC 57 2.0 K149C-(LD-57) ADC-134 Thio Hu Anti-Her27C2 LC K149C- 62 1.7 (LD-62) ADC-135 Thio Hu Anti-Her2 7C2 LC K149C- 631.7 (LD-63) ADC-136 Thio Hu Anti-Her2 7C2 LC K149C- 62 1.8 (LD-62)ADC-137 Thio Hu anti-Her2 7C2 LC K149C- 57 1.9 (LD-57) DAR =drug/antibody ratio average A118C (EU numbering) = A121C (Sequentialnumbering) = A114C (Kabat numbering) Wild-type (“WT”), cysteineengineered mutant antibody (“thio”), light chain (“LC”), heavy chain(“HC”), 6-maleimidocaproyl (“MC”), maleimidopropanoyl (“MP”),valine-citrulline (“val-cit” or “vc”), alanine-phenylalanine(“ala-phe”), p-aminobenzyl (“PAB”), and p- aminobenzyloxycarbonyl(“PABC”)

TABLE 3 Non-cleavable, non-disulfide, comparator ADC: ADC-138 Thio Huanti-Her2 7C2 LC K149C- 1.8 (ethylmaleimide-PNU) ADC-139 Thio HuAnti-CD33 15G15.3 LC K149C- 2.0 (ethylmaleimide-PNU) ADC-140 Thio HuAnti-Her2 7C2 HC A118C- 1.7 (ethylmaleimide-PNU) ADC-141 Trastuzumabemtansine (KADCYLA ®) 3.8

  (ethylmaleimide-PNU) (2S,4S)-4-[[(1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyl-3,4,4a,6,7,9,9a,10a-octahydro-1H-pyrano[1,2]oxazolo[3,4- b][1,4]oxazin-3-yl]oxy]-N-[2-(2,5-dioxopyrrol-1-yl)ethyl]-2,5,12-trihydroxy-7-methoxy-6,11-dioxo-3,4-dihydro-1H- tetracene-2-carboxamide

In Vitro Cell Proliferation Assays

Generally, the cytotoxic or cytostatic activity of an antibody-drugconjugate (ADC) is measured by: exposing mammalian cells having receptorproteins, e.g. HER2, to the antibody of the ADC in a cell culturemedium; culturing the cells for a period from about 6 hours to about 5days; and measuring cell viability. Cell-based in vitro assays were usedto measure viability (proliferation), cytotoxicity, and induction ofapoptosis (caspase activation) of the ADC of the invention.

The in vitro potency of antibody-drug conjugates (ADC) was measured by acell proliferation assay (Example 4). The ADC of the invention showedsurprising and unexpected potency in inhibition of tumor cellproliferation. Potency of the ADC was correlated with target antigenexpression of the cells. The tested conjugates are capable of binding tothe specific antigen expressed on the surface of cells and causing thedeath of those cells in vitro.

The CellTiter-Glo® Luminescent Cell Viability Assay is a commerciallyavailable (Promega Corp., Madison, Wis.), homogeneous assay method basedon the recombinant expression of Coleoptera luciferase (U.S. Pat. No.5,583,024; U.S. Pat. No. 5,674,713; U.S. Pat. No. 5,700,670). This cellproliferation assay determines the number of viable cells in culturebased on quantitation of the ATP present, an indicator of metabolicallyactive cells (Crouch et al (1993) J. Immunol. Meth. 160:81-88; U.S. Pat.No. 6,602,677). The CellTiter-Glo® Assay was conducted in 96 wellformat, making it amenable to automated high-throughput screening (HTS)(Cree et al (1995) AntiCancer Drugs 6:398-404). The homogeneous assayprocedure involves adding the single reagent (CellTiter-Glo® Reagent)directly to cells cultured in serum-supplemented medium. Cell washing,removal of medium and multiple pipetting steps are not required. Thesystem detects as few as 15 cells/well in a 384-well format in 10minutes after adding reagent and mixing. The cells may be treatedcontinuously with ADC, or they may be treated and separated from ADC.Generally, cells treated briefly, i.e. 3 hours, showed the same potencyeffects as continuously treated cells.

The homogeneous “add-mix-measure” format results in cell lysis andgeneration of a luminescent signal proportional to the amount of ATPpresent. The amount of ATP is directly proportional to the number ofcells present in culture. The CellTiterGlo® Assay generates a“glow-type” luminescent signal, produced by the luciferase reaction,which has a half-life generally greater than five hours, depending oncell type and medium used. Viable cells are reflected in relativeluminescence units (RLU). The substrate, Beetle Luciferin, isoxidatively decarboxylated by recombinant firefly luciferase withconcomitant conversion of ATP to AMP and generation of photons.

Cell-based in vitro assays are used to measure viability(proliferation), cytotoxicity, and induction of apoptosis (caspaseactivation) of the ADC of the invention. Generally, the cytotoxic orcytostatic activity of an antibody-drug conjugate (ADC) is measured by:exposing mammalian cells expressing antigen such as Her2 or MUC16polypeptide to ADC in a cell culture medium; culturing the cells for aperiod from about 6 hours to about 5 days; and measuring cell viability.Mammalian cells useful for cell proliferation assays for anti-MUC16 ADCinclude: (1) a MUC16 polypeptide-expressing cell line OVCAR-3; (2) aPC3-derived cell line engineered to stably express a portion of theMUC16 polypeptide on its cell surface (PC3/MUC16); (3) the parental PC3cell line that does not express the MUC16 polypeptide; and (4) a PC3cell line that does not express MUC16 polypeptide but carries the vectorused to drive exogenous MUC16 expression (PC3/neo).

FIG. 1 shows the efficacy of antibody-drug conjugates in a plot ofSK-BR-3 in vitro cell viability at 5 days versus concentrations (μg/ml)of Thio Hu anti-Her2 7C2 HC A118C-(LD-57) 114, Thio Hu anti-Her2 7C2 LCK149C-(LD-57) 109, and Thio Hu anti-CD33 15G15.3 LC K149C-(LD-57) 110.The Her2 antigen is highly expressed in SK-BR-3 cells. Both anti-Her2ADC 114 and 109 showed linear, dose response cell-killing activity. Thelight chain K149C mutant ADC 109 showed the same or slightly more potentcell killing activity than the heavy chain A118C mutant ADC 114.Control, off-target anti-CD33 ADC 110 shows less activity.

FIG. 2 shows the efficacy of antibody-drug conjugates in a plot ofSK-BR-3 in vitro cell viability at 5 days versus concentrations (μg/ml)of Thio Hu anti-Her2 7C2 LC K149C-(LD-59) 116, Thio Hu anti-Her2 7C2 LCK149C-(LD-57) 109, and Thio Hu Anti-CD33 15G15.3 LC K149C-(LD-59) 115.Both anti-Her2 ADC 116 and 109 showed linear, dose response cell-killingactivity. Antibody-drug conjugate 109 with the unsubstituted, lesshindered, ethyl disulfide linker 109 showed the same or slightly morepotent cell killing activity than the methyl substituted, more hindereddisulfide linker ADC-116.

FIG. 6 shows the efficacy of antibody-drug conjugates in a plot ofBJAB.luc in vitro cell viability at 5 days versus concentrations (μg/ml)of Thio Hu Anti-Napi3b 10H1.11.4B LC K149C-(LD-57) 121, Thio Huanti-CD22 10F4v3 LC K149C-(LD-57) 120, Thio Hu Anti-Napi3b 10H1.11.4B LCK149C-(LD-59) 123, Thio Hu anti-CD22 10F4v3 LC K149C-(LD-59) 122, ThioHu Anti-Napi3b 10H1.11.4B LC K149C-(LD-58) 125, Thio Hu anti-CD22 10F4v3LC K149C-(LD-58) 124, Thio Hu Anti-Napi3b 10H1.11.4B LC K149C-(LD-60)127, and Thio Hu anti-CD22 10F4v3 LC K149C-(LD-60) 126.

FIG. 7 shows the efficacy of antibody-drug conjugates in a plot ofWSU-DLCL2 in vitro cell viability at 5 days versus concentrations(μg/ml) of Thio Hu Anti-Napi3b 10H1.11.4B LC K149C-(LD-57) 121, Thio Huanti-CD22 10F4v3 LC K149C-(LD-57) 120, Thio Hu Anti-Napi3b 10H1.11.4B LCK149C-(LD-59) 123, Thio Hu anti-CD22 10F4v3 LC K149C-(LD-59) 122, ThioHu Anti-Napi3b 10H1.11.4B LC K149C-(LD-58) 125, Thio Hu anti-CD22 10F4v3LC K149C-(LD-58) 124, Thio Hu Anti-Napi3b 10H1.11.4B LC K149C-(LD-60)127, and Thio Hu anti-CD22 10F4v3 LC K149C-(LD-60) 126.

In Vivo Efficacy

The in vivo efficacy of antibody-drug conjugates (ADC) of the inventioncan be measured by tumor xenograft studies in mice (Example 22). The invivo efficacy of antibody-drug conjugates (ADC) was measured tumorgrowth inhibition in mice (Example 21). The ADC of the invention showedsurprising and unexpected potency in inhibition of tumor growth.Efficacy of the ADC was correlated with target antigen expression of thetumor cells.

The efficacy of antibody-drug conjugates were measured in vivo byimplanting allografts or xenografts of cancer cells in rodents andtreating the tumors with ADC. Variable results are to be expecteddepending on the cell line, the specificity of antibody binding of theADC to receptors present on the cancer cells, dosing regimen, and otherfactors. The in vivo efficacy of the ADC was measured using a transgenicexplant mouse model expressing moderate to high levels of atumor-associated antigen, such as Her2, MUC16, and CD33. Subjects weretreated once with ADC and monitored over 3-6 weeks to measure the timeto tumor doubling, log cell kill, and tumor shrinkage. Follow updose-response and multi-dose experiments were conducted.

For example, the in vivo efficacy of an anti-HER2 ADC of the inventioncan be measured by a high expressing HER2 transgenic explant mouse model(Phillips et al (2008) Cancer Res. 68:9280-90). An allograft ispropagated from the Fo5 mmtv transgenic mouse which does not respond to,or responds poorly to, HERCEPTIN® therapy. Subjects were treated oncewith ADC at certain dose levels (mg/kg) and placebo buffer control(Vehicle) and monitored over two weeks or more to measure the time totumor doubling, log cell kill, and tumor shrinkage.

FIG. 3 shows the efficacy of antibody-drug conjugates in a plot of thein vivo fitted tumor volume change over time in MMTV-HER2 Fo5 transgenicmammary tumors inoculated into the mammary fat pad of CRL nu/nu miceafter dosing once IV with: Vehicle: Histidine Buffer #8: 20 mM HistidineAcetate, pH 5.5, 240 mM Sucrose, 0.02% PS 20, Thio Hu Anti-Her2 7C2 HCA118C-(LD-51) 107, and Thio Hu Anti-Her2 7C2 LC K149C-(LD-51) 108. ADCwere dosed at 3 mg/kg one time IV at day 0. Both antibody-drugconjugates 107 and 108 showed tumor growth inhibition whereas the lightchain K149C mutant ADC 108 showed greater efficacy than the heavy chainA118C mutant ADC 107.

FIG. 4 shows the efficacy of antibody-drug conjugates in a plot of thein vivo fitted tumor volume change over time in KPL4 tumor model in scidbeige mice inoculated in the thoracic mammary fat pad at a volume of 0.2ml. When tumors reached a mean tumor volume of 100-250 mm3, they weregrouped into 9 groups of 8-10 mice each. A single treatment wasadministered intravenously via the tail vein on Day 0 as follows: (01)Vehicle: Histidine Buffer #8: 20 mM Histidine Acetate, pH 5.5, 240 mMSucrose, 0.02% PS 20, (02) Thio Hu anti-Her2 7C2 LCK149C-(ethylmaleimide) 0.3 mg/kg 138, (03) Thio Hu anti-Her2 7C2 LCK149C-(ethylmaleimide) 1 mg/kg 138, (04) Thio Hu anti-Her2 7C2 LCK149C-(ethylmaleimide) 3 mg/kg 138, (05) Thio Hu anti-Her2 7C2 LCK149C-(LD-57) 0.3 mg/kg 109, (06) Thio Hu anti-Her2 7C2 LC K149C-(LD-57)1 mg/kg 109, (07) Thio Hu anti-Her2 7C2 LC K149C-(LD-57)3 mg/kg 109,(08) Thio Hu Anti-CD33 15G15.3 LC K149C-(ethylmaleimide) 3 mg/kg 139,and (09) Thio Hu anti-CD33 15G15.3 LC K149C-(LD-57) 3 mg/kg 110.

Anti-HER2 7C2 antibody-drug conjugates 138 and 109 showed adose-dependent effect on tumor growth. Non specific anti-CD33antibody-drug conjugates 139 and 110 showed little or no tumor growthinhibition.

FIG. 5 shows the efficacy of antibody-drug conjugates in a plot of thein vivo fitted tumor volume change over time in MMTV-HER2 Fo5 transgenicmammary tumors inoculated into the mammary fat pad of CRL nu/nu miceafter dosing once IV with: (01) Vehicle: Histidine Buffer #8: 20 mMHistidine Acetate, pH 5.5, 240 mM Sucrose, 0.02% PS 20, (02) Thio Huanti-Her2 7C2 LC K149C-(LD-57) 1 mg/kg 109, (03) Thio Hu anti-Her2 7C2LC K149C-(LD-57) 3 mg/kg 109, (04) Thio Hu anti-CD33 15G15.3 LCK149C-(LD-57) 1 mg/kg 110, (05) Thio Hu anti-CD33 15G15.3 LCK149C-(LD-57) 3 mg/kg 110, (06) trastuzumab emtansine 3 mg/kg 141, and(07) trastuzumab emtansine 10 mg/kg 141.

Comparator ADC, trastuzumab emtansine (KADCYLA®, trastuzumab-MCC-DM1(T-DM1) 141, is an antibody-drug conjugate (CAS Reg. No. 139504-50-0;Phillips G. et al. (2008) Cancer Res. 68:9280-90), and has thestructure:

Anti-HER2 7C2 antibody-drug conjugate 109 showed a dose-dependent effecton tumor growth. Non-specific anti-CD33 antibody-drug conjugate 110showed little tumor growth inhibition at 1 mg/kg and modest tumor growthinhibition at 3 mg/kg. Anti-HER2 7C2 antibody-drug conjugate 109 showedmore efficacy than anti-HER2 4D5 trastuzumab emtansine 141 (Phillips G.et al. (2008) Cancer Res. 68:9280-90) at equivalent dose (3 mg/kg) or athigher 10 mg/kg dose.

Pharmaceutical Formulations

Pharmaceutical formulations of therapeutic antibody-drug conjugates(ADC) of the invention are typically prepared for parenteraladministration, i.e. bolus, intravenous, intratumor injection with apharmaceutically acceptable parenteral vehicle and in a unit dosageinjectable form. An antibody-drug conjugate (ADC) having the desireddegree of purity is optionally mixed with pharmaceutically acceptablediluents, carriers, excipients or stabilizers (Remington'sPharmaceutical Sciences (1980) 16th edition, Osol, A. Ed.), in the formof a lyophilized formulation or an aqueous solution.

Antibody-Drug Conjugate Methods of Treatment

It is contemplated that the antibody-drug conjugates (ADC) of thepresent invention may be used to treat various diseases or disorders,e.g. characterized by the overexpression of a tumor antigen. Exemplaryconditions or hyperproliferative disorders include benign or malignantsolid tumors and hematological disorders such as leukemia and lymphoidmalignancies. Others include neuronal, glial, astrocytal, hypothalamic,glandular, macrophagal, epithelial, stromal, blastocoelic, inflammatory,angiogenic and immunologic, including autoimmune, disorders.

The antibody-drug conjugates (ADC) of the invention may be administeredby any route appropriate to the condition to be treated. The ADC willtypically be administered parenterally, i.e. infusion, subcutaneous,intramuscular, intravenous, intradermal, intrathecal and epidural.

Generally, the disease or disorder to be treated is a hyperproliferativedisease such as cancer. Examples of cancer to be treated herein include,but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, andleukemia or lymphoid malignancies. More particular examples of suchcancers include squamous cell cancer (e.g. epithelial squamous cellcancer), lung cancer including small-cell lung cancer, non-small celllung cancer, adenocarcinoma of the lung and squamous carcinoma of thelung, cancer of the peritoneum, hepatocellular cancer, gastric orstomach cancer including gastrointestinal cancer, pancreatic cancer,glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladdercancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectalcancer, endometrial or uterine carcinoma, salivary gland carcinoma,kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer,hepatic carcinoma, anal carcinoma, penile carcinoma, as well as head andneck cancer.

Autoimmune diseases for which the ADC compounds may be used in treatmentinclude rheumatologic disorders (such as, for example, rheumatoidarthritis, Sjögren's syndrome, scleroderma, lupus such as systemic lupuserythematosus (SLE) and lupus nephritis, polymyositis/dermatomyositis,cryoglobulinemia, anti-phospholipid antibody syndrome, and psoriaticarthritis), osteoarthritis, autoimmune gastrointestinal and liverdisorders (such as, for example, inflammatory bowel diseases (e.g.,ulcerative colitis and Crohn's disease), autoimmune gastritis andpernicious anemia, autoimmune hepatitis, primary biliary cirrhosis,primary sclerosing cholangitis, and celiac disease), vasculitis (suchas, for example, ANCA-associated vasculitis, including Churg-Straussvasculitis, Wegener's granulomatosis, and polyarteriitis), autoimmuneneurological disorders (such as, for example, multiple sclerosis,opsoclonus myoclonus syndrome, myasthenia gravis, neuromyelitis optica,Parkinson's disease, Alzheimer's disease, and autoimmunepolyneuropathies), renal disorders (such as, for example,glomerulonephritis, Goodpasture's syndrome, and Berger's disease),autoimmune dermatologic disorders (such as, for example, psoriasis,urticaria, hives, pemphigus vulgaris, bullous pemphigoid, and cutaneouslupus erythematosus), hematologic disorders (such as, for example,thrombocytopenic purpura, thrombotic thrombocytopenic purpura,post-transfusion purpura, and autoimmune hemolytic anemia),atherosclerosis, uveitis, autoimmune hearing diseases (such as, forexample, inner ear disease and hearing loss), Behcet's disease,Raynaud's syndrome, organ transplant, and autoimmune endocrine disorders(such as, for example, diabetic-related autoimmune diseases such asinsulin-dependent diabetes mellitus (IDDM), Addison's disease, andautoimmune thyroid disease (e.g., Graves' disease and thyroiditis)).More preferred such diseases include, for example, rheumatoid arthritis,ulcerative colitis, ANCA-associated vasculitis, lupus, multiplesclerosis, Sjögren's syndrome, Graves' disease, IDDM, pernicious anemia,thyroiditis, and glomerulonephritis.

For the prevention or treatment of disease, the appropriate dosage of anADC will depend on the type of disease to be treated, as defined above,the severity and course of the disease, whether the molecule isadministered for preventive or therapeutic purposes, previous therapy,the patient's clinical history and response to the antibody, and thediscretion of the attending physician. The molecule is suitablyadministered to the patient at one time or over a series of treatments.Depending on the type and severity of the disease, about 1 μg/kg to 15mg/kg (e.g. 0.1-20 mg/kg) of molecule is an initial candidate dosage foradministration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. A typical dailydosage might range from about 1 μg/kg to 100 mg/kg or more, depending onthe factors mentioned above. An exemplary dosage of ADC to beadministered to a patient is in the range of about 0.1 to about 10 mg/kgof patient weight.

Articles of Manufacture

In another embodiment of the invention, an article of manufacture, or“kit”, containing materials useful for the treatment of the disordersdescribed above is provided. The article of manufacture comprises acontainer and a label or package insert on or associated with thecontainer. Suitable containers include, for example, bottles, vials,syringes, blister pack, etc. The containers may be formed from a varietyof materials such as glass or plastic. The container holds anantibody-drug conjugate (ADC) composition which is effective fortreating the condition and may have a sterile access port (for examplethe container may be an intravenous solution bag or a vial having astopper pierceable by a hypodermic injection needle). At least oneactive agent in the composition is an ADC. The label or package insertindicates that the composition is used for treating the condition ofchoice, such as cancer. Alternatively, or additionally, the article ofmanufacture may further comprise a second (or third) containercomprising a pharmaceutically-acceptable buffer, such as bacteriostaticwater for injection (BWFI), phosphate-buffered saline, Ringer's solutionand dextrose solution. It may further include other materials desirablefrom a commercial and user standpoint, including other buffers,diluents, filters, needles, and syringes.

EXAMPLES Example 1 Synthesis of Anthracycline Disulfide, Linker-Drug(LD) Intermediates (Table 1) LD-51: Synthesis of(2S,4S)-4-[[(1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyl-3,4,4a,6,7,9,9a,10a-octahydro-1H-pyrano[1,2]oxazolo[3,4-b][1,4]oxazin-3-yl]oxy]-2,5,12-trihydroxy-7-methoxy-6,11-dioxo-N-[2-(2-pyridyldisulfanyl)ethyl]-3,4-dihydro-1H-tetracene-2-carboxamide

Following Example 3 of U.S. Pat. No. 8,389,697, to a solution ofPNU-159682 (15.3 mg, 0.02038 mmol), prepared as reported in WO1998/02446 and Example 1 of U.S. Pat. No. 8,470,984, in 3 ml of methanoland 2 ml of H₂O, a solution of NaIO₄ (5.1 mg, 0.0238 mmol) in 1 ml ofH₂O was added. The reaction mixture was stirred at room temperature for3 hours, until no starting material was detectable (TLC and HPLCanalysis). _The solvents were removed under reduced pressure and thecrude red solid (2S,4S)-2,5,12-trihydroxy-7-methoxy-4-{[(1S,3R,4 aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4′,3′:4,5][1,3]oxazolo[2,3-c][1,4]oxazin-3-yl]oxy}-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2-carboxylicacid 51a was used without further purifications in the next step. MS(ESI): 628 [M+H]⁺.

To a solution of the crude intermediate 51a in anhydrous dichloromethaneunder argon atmosphere, was added anhydrous triethylamine, TBTU(O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate,also called: N,N,N′,N′-Tetramethyl-O-(benzotriazol-1-yl)uroniumtetrafluoroborate, CAS No. 125700-67-6, Sigma-Aldrich B-2903), andN-hydroxysuccinimide to form the intermediate NHS ester of 51a.Alternatively, other coupling reagents such as DCC or EDC can be used.After one hour, 2-(pyridin-2-yl disulfanyl)ethanamine hydrochloride (CASNo. 106139-15-5) was added. The reaction mixture was stirred at roomtemperature for 30 min, until disappearance of the starting material(HPLC-MS analysis). The solvent was evaporated under vacuum and theresidue was then purified by flash column chromatography on silica gel,affording 51. MS (ESI): 796.88 [M+H]⁺.

LD-52: Synthesis of 2-(2-pyridyldisulfanyl)ethylN-[2-[[2-[(2S,4S)-4-[[(1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyl-3,4,4a,6,7,9,9a,10a-octahydro-1H-pyrano[1,2]oxazolo[3,4-b][1,4]oxazin-3-yl]oxy]-2,5,12-trihydroxy-7-methoxy-6,11-dioxo-3,4-dihydro-1H-tetracen-2-yl]-2-oxo-ethoxy]carbonyl-methyl-amino]ethyl]-N-methyl-carbamate

[2-[(2S,4 S)-4-[[(1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyl-3,4,4a,6,7,9,9a,10a-octahydro-1H-pyrano[1,2]oxazolo[3,4-b][1,4]oxazin-3-yl]oxy]-2,5,12-trihydroxy-7-methoxy-6,11-dioxo-3,4-dihydro-1H-tetracen-2-yl]-2-oxo-ethyl]N-methyl-N-[2-(methylamino)ethyl]carbamate 52a was prepared fromPNU-159682, with triphosgene (Cl₃CO)₂C═O, andN1,N2-dimethylethane-1,2-diamine.

1,2-Di(pyridin-2-yl)disulfane and 2-mercaptoethanol were reacted inpyridine and methanol at room temperature to give2-(pyridin-2-yldisulfanyl)ethanol. Acylation with 4-nitrophenylcarbonochloridate in triethylamine and acetonitrile gave 4-nitrophenyl2-(pyridin-2-yldisulfanyl)ethyl carbonate 52b. Intermediate 52a wasreacted with 52b to form LD-52.

LD-53: Synthesis of[2-methyl-2-(2-pyridyldisulfanyl)propyl]N-[2-[[2-[(2S,4 S)-4-[[(1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyl-3,4,4a,6,7,9,9a,10a-octahydro-1H-pyrano[1,2]oxazolo[3,4-b][1,4]oxazin-3-yl]oxy]-2,5,12-trihydroxy-7-methoxy-6,11-dioxo-3,4-dihydro-1H-tetracen-2-yl]-2-oxo-ethoxy]carbonyl-methyl-amino]ethyl]-N-methyl-carbamate

Following the procedures for LD-52, intermediate 52a was acylated with2-methyl-2-(pyridin-2-yldisulfanyl)propyl 4-nitrophenyl carbonate togive LD-53.

LD-54: Synthesis of(2S,4S)-4-[[(1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyl-3,4,4a,6,7,9,9a,10a-octahydro-1H-pyrano[1,2]oxazolo[3,4-b][1,4]oxazin-3-yl]oxy]-2,5,12-trihydroxy-7-methoxy-N-[2-methyl-2-(2-pyridyldisulfanyl)propyl]-6,11-dioxo-3,4-dihydro-1H-tetracene-2-carboxamide

Following the procedures for LD-51, crude intermediate 51a in anhydrousdichloromethane under argon atmosphere, was reacted with anhydroustriethylamine, TBTU, and2-methyl-2-(pyridin-2-yldisulfanyl)propan-1-amine to afford LD-54.

LD-55: Synthesis of(2S,4S)-4-[[(1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyl-3,4,4a,6,7,9,9a,10a-octahydro-1H-pyrano[1,2]oxazolo[3,4-b][1,4]oxazin-3-yl]oxy]-2,5,12-trihydroxy-7-methoxy-N-[2-methyl-2-[(5-nitro-2-pyridyl)disulfanyl]propyl]-6,11-dioxo-3,4-dihydro-1H-tetracene-2-carboxamide

Following the procedures for LD-60, 5-nitropyridine-2-thiol 60a wasadded to a solution of LD-54 in DMF to give LD-55.

LD-56: Synthesis of 2-(2-pyridyldisulfanyl)propylN-methyl-N-[2-[methyl-[2-oxo-2-[2,5,12-trihydroxy-7-methoxy-4-[(9-methoxy-1-methyl-3,4,4a,6,7,9,9a,10a-octahydro-1H-pyrano[1,2]oxazolo[3,4-b][1,4]oxazin-3-yl)oxy]-6,11-dioxo-3,4-dihydro-1H-tetracen-2-yl]ethoxy]carbonyl-amino]ethyl]carbamate

Following the procedures for LD-52, intermediate 52a was acylated with4-nitrophenyl 2-(pyridin-2-yldisulfanyl)propyl carbonate to give LD-56.

LD-57: Synthesis of(2S,4S)-4-[[(1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyl-3,4,4a,6,7,9,9a,10a-octahydro-1H-pyrano[1,2]oxazolo[3,4-b][1,4]oxazin-3-yl]oxy]-2,5,12-trihydroxy-7-methoxy-N-[2-[(5-nitro-2-pyridyl)disulfanyl]ethyl]-6,11-dioxo-3,4-dihydro-1H-tetracene-2-carboxamide

To a mixture of 1,2-bis(5-nitropyridin-2-yl)disulfane 57a (1.0 g, 3.22mmol) in anhydrous DMF/MeOH (25 mL/25 mL) was added HOAc (0.1 mL),followed by 2-aminoethanethiol hydrochloride 57b (183 mg, 1.61 mmol).After the reaction mixture was stirred at r.t. overnight, it wasconcentrated under vacuum to remove the solvent, and the residue waswashed with DCM (30 mL×4) to afford2-((5-nitropyridin-2-yl)disulfanyl)ethanamine hydrochloride 57c as paleyellow solid (300 mg, 69.6%). ¹H NMR (400 MHz, DMSO-d₆) δ 9.28 (d, J=2.4Hz, 1H), 8.56 (dd, J=8.8, 2.4 Hz, 1H), 8.24 (s, 4H), 8.03 (d, J=8.8 Hz,1H), 3.15-3.13 (m, 2H), 3.08-3.06 (m, 2H)

Intermediate 51a was prepared as above. Alternatively, to a suspensionof PNU-159682 (50 mg, 0.078 mmol) in MeOH/H₂O (5 mL/5 mL) was added asolution of NaIO₄ (17 mg, 0.078 mmol) in H₂O (2 mL). The heterogeneousreaction mixture was vigorously stirred at r.t. for 4 h in the dark.Then the mixture was concentrated under vacuum, and the residue waspurified by column chromatography on silica gel (DCM/MeOH=100/1 to 10/1)to afford intermediate 51a as purple solid (40 mg, 80%).

To a solution of intermediate 51a (40 mg, 0.064 mmol) in anhydrous DCM(5 mL) was added DIEA (41 mg, 0.32 mmol) and HATU (isO-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate, 73 mg, 0.19 mmol). After the mixture was stirred atr.t. for 0.5 h, intermediate 57c (51 mg, 0.19 mmol) was added. Theresulting mixture was stirred at r.t. for another 4 h. The mixture waspurified by prep-TLC (DCM/MeOH=20/1) to afford LD-57 as purple solid (18mg, 33.3%). LCMS (ESI): RT=0.890 min, M+H⁺=841.2., Method=5-95AB/1.5min. ¹H NMR (400 MHz, CDCl₃) δ 13.94 (s, 1H), 13.30 (s, 1H), 9.33 (d,J=2.0 Hz, 1H), 8.40 (dd, J=9.2, 2.4 Hz, 1H), 8.22 (t, J=6.0 Hz, 1H),8.04 (J=7.6 Hz, 1H), 7.80-7.76 (m, 2H), 7.40 (d, J=8.8 Hz, 1H), 5.52 (d,J=5.6 Hz, 1H), 5.34 (s, 1H), 5.30 (s, 1H), 5.22 (s, 1H), 4.69 (d, J=1.6Hz, 1H), 4.47 (d, J=0.8 Hz, 1H), 4.10 (s, 3H), 4.07-4.01 (m, 2H), 3.91(t, J=9.6 Hz, 1H), 3.74 (s, 2H), 3.70-3.55 (m, 4H), 3.45 (s, 3H),3.41-3.38 (m, 1H), 3.25 (d, J=2.4 Hz, 2H), 3.05 (t, J=6.0 Hz, 2H),2.83-2.80 (m, 1H), 2.73-2.68 (m, 1H), 2.52-2.48 (m, 1H), 2.41-2.36 (m,1H), 2.05-1.98 (s, 1H), 1.81-1.75 (m, 1H), 1.36 (d, J=6.0 Hz, 3H)

LD-58: Synthesis of 2-[(5-nitro-2-pyridyl)disulfanyl]ethylN-[2-[[2-[(2S,4S)-4-[[(1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyl-3,4,4a,6,7,9,9a,10a-octahydro-1H-pyrano[1,2]oxazolo[3,4-b][1,4]oxazin-3-yl]oxy]-2,5,12-trihydroxy-7-methoxy-6,11-dioxo-3,4-dihydro-1H-tetracen-2-yl]-2-oxo-ethoxy]carbonyl-methyl-amino]ethyl]-N-methyl-carbamate

A solution of 1,2-bis(5-nitropyridin-2-yl)disulfane 57a (9.6 g, 30.97mmol) and 2-mercaptoethanol (1.21 g, 15.49 mmol) in anhydrous DCM/CH₃OH(250 mL/250 mL) was stirred at r.t. under N2 for 24 h. After the mixturewas concentrated under vacuum, and the residue was diluted with DCM (300mL). MnO₂ (10 g) was added and the mixture was stirred at r.t. foranother 0.5 h. The mixture was purified by column chromatography onsilica gel (DCM/MeOH=100/1 to 100/1) to afford2-((5-nitropyridin-2-yl)disulfanyl)ethanol 58a (2.2 g, 61.1%) as brownoil. ¹H NMR (400 MHz, CDCl₃) δ 9.33 (d, J=2.8 Hz, 1H), 8.38-8.35 (dd,J=9.2, 2.8 Hz, 1H), 7.67 (d, J=9.2 Hz, 1H), 4.10 (t, J=7.2 Hz, 1H),3.81-3.76 (q, 2H), 3.01 (t, J=5.2 Hz, 2H).

To a solution of 58a (500 mg, 2.15 mmol) in anhydrous DMF (10 mL) wasadded DIEA (834 mg, 6.45 mmol), followed by PNP carbonate(bis(4-nitrophenyl) carbonate, 1.31 g, 4.31 mmol). The reaction solutionwas stirred at r.t for 4 h and the mixture was purified by prep-HPLC(FA) to afford 4-nitrophenyl 2-((5-nitropyridin-2-yl)disulfanyl)ethylcarbonate 58b (270 mg, 33.1%) as light brown oil. ¹H NMR (400 MHz,CDCl₃) δ 9.30 (d, J=2.4 Hz, 1H), 8.43-8.40 (dd, J=8.8, 2.4 Hz, 1H),8.30-8.28 (m, 2H), 7.87 (d, J=8.8 Hz, 1H), 7.39-7.37 (m, 2H), 4.56 (t,J=6.4 Hz, 2H), 3.21 (t, J=6.4 Hz, 2H).

[2-[(2S,4S)-4-[[(1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyl-3,4,4a,6,7,9,9a,10a-octahydro-1H-pyrano[1,2]oxazolo[3,4-b][1,4]oxazin-3-yl]oxy]-2,5,12-trihydroxy-7-methoxy-6,11-dioxo-3,4-dihydro-1H-tetracen-2-yl]-2-oxo-ethyl]N-methyl-N-[2-(methylamino)ethyl]carbamate 52a was prepared from PNU viadeprotection of Bpoc-protected 58c where Bpoc is2-(p-biphenylyl)-2-propyloxycarbonyl. To a solution of 58c (25 mg, 0.025mmol), where the amine is protected with 2-(4-biphenylyl)-prop-2-yl4′-methoxycarbonylphenyl carbonate (BPOC reagent, Sigma-Aldrich, CASReg. No. 31140-37-1) in anhydrous DCM (4 mL) was added a solution ofdichloroacetic acid (Cl₂CHCOOH, 65 mg, 0.50 mmol) in anhydrous DCM (1mL). The reaction solution was stirred at r.t for 2 h. The solution wasconcentrated under vacuum and the residue was washed with methyl,tert-butyl ether (MTBE, 10 mL×2) to afford 52a (25 mg, 100%) as redsolid.

To a solution of 52a (25 mg, 0.025 mmol) and 4-nitrophenyl2-((5-nitropyridin-2-yl)disulfanyl)ethyl carbonate 58b (30 mg, 0.075mmol) in anhydrous DMF (3 mL) was added TEA (triethylamine, 25 mg, 0.25mmol). After the solution was stirred at r.t. overnight, it wasconcentrated under vacuum and the residue was purified by prep-TLC(DCM/MeOH=20/1) to afford LD-58 (12 mg, 48.0%) as red solid. LCMS (ESI):RT=0.921 min, M+Na⁺=1036.2. ¹H NMR (400 MHz, CDCl₃) δ 13.88 (s, 1H),13.24 (d, J=4.0 Hz, 1H), 9.26-9.25 (m, 1H), 8.40 (d, J=6.0 Hz, 1H), 8.01(d, J=7.6 Hz, 1H), 7.89 (d, J=8.8 Hz, 1H), 7.77 (t, J=8.0 Hz, 1H), 7.38(d, J=8.4 Hz, 1H), 5.47 (s, 1H), 5.30-5.23 (m, 2H), 5.15-5.11 (m, 1H),4.92-4.87 (m, 1H), 4.70 (s, 1H), 4.47 (s, 1H), 4.35 (s, 2H), 4.10-4.03(m, 4H), 3.92-3.87 (m, 1H), 3.59-3.40 (m, 8H), 3.25-3.22 (m, 1H),3.12-3.10 (m, 2H), 3.04 (s, 2H), 3.98 (t, J=8.8 Hz, 4H), 2.84-2.82 (m,2H), 2.60 (d, J=14.8 Hz, 1H), 2.07-1.98 (m, 1H), 1.76-1.72 (m, 1H), 1.41(d, J=5.6 Hz, 3H).

LD-59: Synthesis of2,5,12-trihydroxy-7-methoxy-4-[(9-methoxy-1-methyl-3,4,4a,6,7,9,9a,10a-octahydro-1H-pyrano[1,2]oxazolo[3,4-b][1,4]oxazin-3-yl)oxy]-N-[2-[(5-nitro-2-pyridyl)disulfanyl]propyl]-6,11-dioxo-3,4-dihydro-1H-tetracene-2-carboxamide

To a stirred solution of 1-aminopropan-2-ol 59a (10 g, 133 mmol) in MeOH(360 mL) and H₂O (40 mL) was added Boc₂O (37 g, 169 mmol). After thereaction mixture was stirred at r.t. for 5 h, it was concentrated andpurified by chromatography (EtOAc/PE=10%-50%) to give tert-butyl2-hydroxypropylcarbamate 59b as a colorless oil (19.8 g, yield: 85%).

To a stirred solution of 59b (10 g, 57 mmol) and Et₃N (17 g, 171 mmol)in DCM (130 mL) was added a solution of MSCl (methanesulfonyl chloride,13 g, 114 mmol). After the reaction mixture was stirred at r.t. for 4 h,it was washed with ice water (200 mL×3) and brine (200 mL). The organiclayer was concentrated to give 1-(tert-butoxycarbonylamino)propan-2-ylmethanesulfonate 59c as a red oil (12 g, yield: 83%).

To a stirred solution of 59c (6 g, 23.7 mmol) in acetone (70 mL) wasadded a solution of potassium thioacetate (potassium ethanethioate, 5.4g, 47.3 mmol) in H₂O (100 mL). The reaction mixture was stirred at 60°C. for 12 h. The mixture was concentrated and extracted with DCM (200ml×2). The combined organic layers were concentrated and purified bychromatography to give S-1-(tert-butoxycarbonylamino)propan-2-ylethanethioate 59d as a red solid (1.1 g, yield: 20%). ¹H NMR (400 MHz,CDCl3-d) δ 1.30 (d, J=7.09 Hz, 3H) 1.44 (s, 9H) 2.33 (s, 3H) 3.16-3.42(m, 2H) 3.58-3.71 (m, 1H)

To a stirred solution of 59d (500 mg, 2.15 mmol) in MeOH (5 mL) wasadded HCl/MeOH (10 mL) dropwise. After the reaction mixture was stirredat r.t. for 3 h, it was concentrated to give 1-aminopropane-2-thiolhydrochloride 59e, used directly in the next step.

To a solution of 57a (1.33 g, 4.3 mmol) in DCM (35 mL) was added asolution of 59e (273 mg, 2.15 mmol). The mixture was stirred at 15° C.for 12 h. MnO₂ (374.1 mg, 4.3 mmol) was added to the mixture and stirredat 15° C. for 10 min. The solid was washed with DCM (100 mL) and MeOH(30 mL×3). The solution was concentrated to give2-((5-nitropyridin-2-yl)disulfanyl)propan-1-amine 59f as a yellow solid(300 mg, 57%). LCMS (ESI): RT=0.546 min, M+H⁺=245.7.

To a solution of intermediate 51a (25 mg, 0.040 mmol) in anhydrousDCM/DMF (5 mL) was added DIEA (diisopropylethylamine, 26 mg, 0.20 mmol)and HATU (is O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate, 46 mg, 0.12 mmol). After the mixture was stirred atr.t for 0.5 h, 59f (34 mg, 0.12 mmol) was added. The resulting mixturewas stirred at r.t for another 4 h. The mixture was diluted with DCM (30mL), and washed with brine (15 mL×3). The DCM layer was dried (Na₂SO₄),filtered, and concentrate. The residue was purified by prep-TLC(DCM/MeOH=20/1) to afford LD-59 (7 mg, 20.6%) as purple solid. LCMS(ESI): RT=0.783 min, M+H⁺=855.2. ¹H NMR (400 MHz, CDCl₃) δ 13.94 (d,J=3.6 Hz, 1H), 13.32 (d, J=4.8 Hz, 1H), 9.33 (s, 1H), 8.63-8.47 (m, 1H),8.39-8.35 (m, 1H), 8.04 (d, J=7.6 Hz, 1H), 7.78 (J=8.0 Hz, 1H),7.74-7.71 (dd, J=8.8, 5.6 Hz, 1H), 7.39 (d, J=8.8 Hz, 1H), 5.52 (d,J=5.2 Hz, 1H), 5.35 (s, 1H), 5.27 (d, J=4.8 Hz, 1H), 4.68 (d, J=4.4 Hz,1H), 4.46 (d, J=8.0 Hz, 1H), 4.10 (s, 3H), 4.06 (d, J=7.6 Hz, 1H), 3.92(t, J=7.8 Hz, 2H), 3.71-3.67 (m, 1H), 3.59-3.55 (m, 2H), 3.45 (d, J=6.4Hz, 3H), 3.41-3.36 (m, 1H), 3.28-3.22 (m, 3H), 2.84-2.60 (m, 2H),2.53-2.37 (m, 2H), 2.06-1.94 (m, 1H), 1.81-1.77 (m, 1H), 1.40-1.38 (dd,J=6.4, 2.4 Hz, 3H), 1.35 (t, J=6.0 Hz, 3H)

LD-60: Synthesis of[2-oxo-2-[2,5,12-trihydroxy-7-methoxy-4-[(9-methoxy-1-methyl-3,4,4a,6,7,9,9a,10a-octahydro-1H-pyrano[1,2]oxazolo[3,4-b][1,4]oxazin-3-yl)oxy]-6,11-dioxo-3,4-dihydro-1H-tetracen-2-yl]ethyl]N-methyl-N-[2-[methyl-[2-[(5-nitro-2-pyridyl)disulfanyl]propoxycarbonyl]amino]ethyl]carbamate

To a solution of 2-(2-pyridyldisulfanyl)propylN-methyl-N-[2-[methyl-[2-oxo-2-[2,5,12-trihydroxy-7-methoxy-4-[(9-methoxy-1-methyl-3,4,4a,6,7,9,9a,10a-octahydro-1H-pyrano[1,2]oxazolo[3,4-b][1,4]oxazin-3-yl)oxy]-6,11-dioxo-3,4-dihydro-1H-tetracen-2-yl]ethoxy]carbonyl-amino]ethyl]carbamateLD-56 (10 mg, 10.17 umol) in DMF (1 mL) was added a solution of5-nitropyridine-2-thiol 60a (15.89 mg, 101.72 umol) at 20° C. Thereaction mixture was stirred at 20° C. for 2 h. The reaction mixture wasdiluted with DCM (10 mL) and then washed with water (5 mL), aq.NaHCO₃ (5mL) and brine (5 mL). The organic layer was dried and concentrated. Theresidue was purified by prep-TLC (DCM:MeOH=25:1) to give pure LD-60 (7mg, 66.94%) as a red solid. LCMS: (10-80, CD, 7.0 min), 3.782 min,Ms=1028.2 (M+1); ¹H NMR (400 MHz, CDCl₃) δ 13.83 (s, 1H), 13.19 (s, 1H),9.18 (s, 1H), 8.31 (d, 1H, J=7.6 Hz), 7.97 (d, 1H, J=8.0 Hz), 7.86 (d,1H, J=8.4 Hz), 7.73 (t, 1H, J=8.4 Hz), 7.33 (d, 1H, J=8.4 Hz), 5.41 (s,1H), 5.29-5.05 (m, 3H), 4.89-4.75 (m, 1H), 4.64 (s, 1H), 4.40 (s, 1H),4.13 (s, 2H), 4.09-3.80 (m, 6H), 3.58-3.12 (m, 12H), 3.05-2.85 (m, 7H),2.78-2.42 (m, 3H), 2.12-1.85 (m, 2H), 1.72-1.55 (m, 1H), 1.38-1.25 (m,6H)

LD-61: Synthesis of2,5,12-trihydroxy-7-methoxy-4-[(9-methoxy-1-methyl-3,4,4a,6,7,9,9a,10a-octahydro-1H-pyrano[1,2]oxazolo[3,4-b][1,4]oxazin-3-yl)oxy]-6,11-dioxo-N-[2-(2-pyridyldisulfanyl)propyl]-3,4-dihydro-1H-tetracene-2-carboxamide

Following the procedures for LD-51, crude intermediate 51a in anhydrousdichloromethane under argon atmosphere, was reacted with anhydroustriethylamine, TBTU, and 2-(pyridin-2-yldisulfanyl)propan-1-amine toafford LD-61.

LD-62: Synthesis of(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4′,3′:4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-N—((R)-2-((5-nitropyridin-2-yl)disulfanyl)propyl)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2-carboxamide

Following the procedures for LD-59, LD-62 was prepared.

LD-63: Synthesis of(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4′,3′:4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-N—((S)-2-((5-nitropyridin-2-yl)disulfanyl)propyl)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2-carboxamide

Following the procedures for LD-59, LD-63 was prepared.

Example 2 Preparation of Cysteine Engineered Antibodies for Conjugationby Reduction and Reoxidation

Under certain conditions, the cysteine engineered antibodies may be madereactive for conjugation with linker-drug intermediates of theinvention, such as those in Table 1, by treatment with a reducing agentsuch as DTT (Cleland's reagent, dithiothreitol) or TCEP(tris(2-carboxyethyl)phosphine hydrochloride; Getz et al (1999) Anal.Biochem. Vol 273:73-80; Soltec Ventures, Beverly, Mass.) in 50 mM TrispH 7.5 with 2 mM EDTA for 3 hrs at 37° C. or overnight at roomtemperature. Full length, cysteine engineered monoclonal antibodies(THIOMAB™) expressed in CHO cells (Gomez et al (2010) Biotechnology andBioeng. 105(4):748-760; Gomez et al (2010) Biotechnol. Prog.26:1438-1445) were reduced, for example with about a 50 fold excess ofDTT overnight at room temperature to reduce disulfide bonds which mayform between the newly introduced cysteine residues and the cysteinepresent in the culture media. The reduced THIOMAB™ was diluted andloaded onto a HiTrap S column in 10 mM sodium acetate, pH 5, and elutedwith PBS containing 0.3M sodium chloride. Alternatively, the antibodywas acidified by addition of 1/20^(th) volume of 10% acetic acid,diluted with 10 mM succinate pH 5, loaded onto the column and thenwashed with 10 column volumes of succinate buffer. The column was elutedwith 50 mM Tris pH7.5, 2 mM EDTA.

Light chain amino acids are numbered according to Kabat (Kabat et al.,Sequences of proteins of immunological interest, (1991) 5th Ed., US Deptof Health and Human Service, National Institutes of Health, Bethesda,Md.). Heavy chain amino acids are numbered according to the EU numberingsystem (Edelman et al (1969) Proc. Natl. Acad. of Sci. 63(1):78-85),except where noted as the Kabat system. Single letter amino acidabbreviations are used.

Full length, cysteine engineered monoclonal antibodies (THIOMAB™)expressed in CHO cells bear cysteine adducts (cystines) orglutathionylated on the engineered cysteines due to cell cultureconditions. To liberate the reactive thiol groups of the engineeredcysteines, the THIOMAB™ are dissolved in 500 mM sodium borate and 500 mMsodium chloride at about pH 8.0 and reduced with about a 50-100 foldexcess of 1 mM TCEP (tris(2-carboxyethyl)phosphine hydrochloride (Getzet al (1999) Anal. Biochem. Vol 273:73-80; Soltec Ventures, Beverly,Mass.) for about 1-2 hrs at 37° C. Alternatively, DTT can be used asreducing agent. The formation of inter-chain disulfide bonds wasmonitored either by non-reducing SDS-PAGE or by denaturing reverse phaseHPLC PLRP column chromatography. The reduced THIOMAB™ is diluted andloaded onto a HiTrap SP FF column in 10 mM sodium acetate, pH 5, andeluted with PBS containing 0.3M sodium chloride, or 50 mM Tris-Cl, pH7.5 containing 150 mM sodium chloride.

Disulfide bonds were reestablished between cysteine residues present inthe parent Mab by carrying out reoxidation. The eluted reduced THIOMAB™is treated with 15× or 2 mM dehydroascorbic acid (dhAA) at pH 7 forabout 3 hours or for about 3 hrs in 50 mM Tris-Cl, pH 7.5, or with 200nM to 2 mM aqueous copper sulfate (CuSO₄) at room temperature overnight.Other oxidants, i.e. oxidizing agents, and oxidizing conditions, whichare known in the art may be used. Ambient air oxidation may also beeffective. This mild, partial reoxidation step forms intrachaindisulfides efficiently with high fidelity. The buffer is exchanged byelution over Sephadex G25 resin and eluted with PBS with 1 mM DTPA. Thethiol/Ab value is checked by determining the reduced antibodyconcentration from the absorbance at 280 nm of the solution and thethiol concentration by reaction with DTNB (Aldrich, Milwaukee, Wis.) anddetermination of the absorbance at 412 nm.

Liquid chromatography/Mass Spectrometric Analysis was performed on a TSQQuantum Triple quadrupole™ mass spectrometer with extended mass range(Thermo Electron, San Jose Calif.). Samples were chromatographed on aPRLP-S®, 1000 A, microbore column (50 mm×2.1 mm, Polymer Laboratories,Shropshire, UK) heated to 75° C. A linear gradient from 30-40% B(solvent A: 0.05% TFA in water, solvent B: 0.04% TFA in acetonitrile)was used and the eluent was directly ionized using the electrospraysource. Data were collected by the Xcalibur® data system anddeconvolution was performed using ProMass® (Novatia, LLC, New Jersey).Prior to LC/MS analysis, antibodies or drug conjugates (50 micrograms)were treated with PNGase F (2 units/ml; PROzyme, San Leandro, Calif.)for 2 hours at 37° C. to remove N-linked carbohydrates.

Hydrophobic Interaction Chromatography (HIC) samples were injected ontoa Butyl HIC NPR column (2.5 micron particle size, 4.6 mm×3.5 cm) (TosohBioscience) and eluted with a linear gradient from 0 to 70% B at 0.8ml/min (A: 1.5 M ammonium sulfate in 50 mM potassium phosphate, pH 7, B:50 mM potassium phosphate pH 7, 20% isopropanol). An Agilent 1100 seriesHPLC system equipped with a multi wavelength detector and Chemstationsoftware was used to resolve and quantitate antibody species withdifferent ratios of drugs per antibody.

Example 3 Conjugation of Linker-Drug Intermediates to Antibodies

After the reduction and reoxidation procedures of Example 2, thecysteine-engineered antibody (THIOMAB™) is dissolved in PBS (phosphatebuffered saline) buffer and chilled on ice. An excess, from about 1.5molar to 20 equivalents of a linker-drug intermediate, including but notlimited to LD-51 to LD-61 in Table 1, with a thiol-reactive pyridyldisulfide group, is dissolved in DMSO, diluted in acetonitrile andwater, and added to the chilled reduced, reoxidized antibody in PBS.Typically the linker-drug is added from a DMSO stock at a concentrationof about 20 mM in 50 mM Tris, pH 8, to the antibody and monitored untilthe reaction is complete from about 1 to about 24 hours as determined byLC-MS analysis of the reaction mixture. When the reaction is complete,an excess of maleimide is added to quench the reaction and cap anyunreacted antibody thiol groups. The conjugation mixture may be loadedand eluted through a HiTrap SP FF column to remove excess drug-linkerintermediate and other impurities. The reaction mixture is concentratedby centrifugal ultrafiltration and the cysteine engineered antibody drugconjugate is purified and desalted by elution through G25 resin in PBS,filtered through 0.2 μm filters under sterile conditions, and frozen forstorage.

For example, the crude antibody-drug conjugate (ADC) is applied to acation exchange column after dilution with 20 mM sodium succinate, pH 5.The column was washed with at least 10 column volumes of 20 mM sodiumsuccinate, pH 5, and the antibody was eluted with PBS. The antibody drugconjugates were formulated into 20 mM His/acetate, pH 5, with 240 mMsucrose using gel filtration columns. The antibody-drug conjugates werecharacterized by UV spectroscopy to determine protein concentration,analytical SEC (size-exclusion chromatography) for aggregation analysisand LC-MS before and after treatment with Lysine C endopeptidase.

Size exclusion chromatography is performed using a Shodex KW802.5 columnin 0.2M potassium phosphate pH 6.2 with 0.25 mM potassium chloride and15% IPA at a flow rate of 0.75 ml/min. Aggregation state of theconjugate was determined by integration of eluted peak area absorbanceat 280 nm.

LC-MS analysis may be performed using an Agilent QTOF 6520 ESIinstrument. As an example, the antibody-drug conjugate is treated with1:500 w/w Endoproteinase Lys C (Promega) in Tris, pH 7.5, for 30 min at37° C. The resulting cleavage fragments are loaded onto a 1000A(Angstrom), 8 μm (micron) PLRP-S (highly cross-linked polystyrene)column heated to 80° C. and eluted with a gradient of 30% B to 40% B in5 minutes. Mobile phase A was H₂O with 0.05% TFA and mobile phase B wasacetonitrile with 0.04% TFA. The flow rate was 0.5 ml/min. Proteinelution was monitored by UV absorbance detection at 280 nm prior toelectrospray ionization and MS analysis. Chromatographic resolution ofthe unconjugated Fc fragment, residual unconjugated Fab and drugged Fabwas usually achieved. The obtained m/z spectra were deconvoluted usingMass Hunter™ software (Agilent Technologies) to calculate the mass ofthe antibody fragments.

By these procedures, cysteine engineered, antibody drug conjugates101-117 of Table 2 were prepared.

Example 4 In Vitro Cell Proliferation Assay

Efficacy of ADC was measured by a cell proliferation assay employing thefollowing protocol (CELLTITER GLO™ Luminescent Cell Viability Assay,Promega Corp. Technical Bulletin TB288; Mendoza et al (2002) Cancer Res.62:5485-5488):

-   1. An aliquot of 100 μl of cell culture containing about 10⁴ cells    (SKBR-3, BT474, MCF7 or MDA-MB-468) in medium was deposited in each    well of a 96-well, opaque-walled plate.-   2. Control wells were prepared containing medium and without cells.-   3. ADC was added to the experimental wells and incubated for 3-5    days.-   4. The plates were equilibrated to room temperature for    approximately 30 minutes.-   5. A volume of CELLTITER GLO™ Reagent equal to the volume of cell    culture medium present in each well was added.-   6. The contents were mixed for 2 minutes on an orbital shaker to    induce cell lysis.-   7 The plate was incubated at room temperature for 10 minutes to    stabilize the luminescence signal.-   8. Luminescence was recorded and reported in graphs as RLU=relative    luminescence units.-   Data are plotted as the mean of luminescence for each set of    replicates, with standard deviation error bars. The protocol is a    modification of the CELLTITER GLO™ Luminescent Cell-   Media: SK-BR-3 grow in 50/50/10% FBS/glutamine/250 μg/mL G-418    OVCAR-3 grow in RPMI/20% FBS/glutamine

Example 5 Tumor Growth Inhibition, In Vivo Efficacy in High ExpressingHER2 Transgenic Explant Mice

Tumors were established and allowed to grow to 150-200 mm³ in volume (asmeasured using calipers) before a single treatment on day 0. Tumorvolume was measured using calipers according to the formula: V(mm³)=0.5A×B², where A and B are the long and short diameters,respectively. Mice were euthanized before tumor volume reached 3000 mm³or when tumors showed signs of impending ulceration. Data collected fromeach experimental group (10 mice per group) were expressed as mean±SE.

The Fo5 mouse mammary tumor model was employed to evaluate the in vivoefficacy of antibody-drug conjugates of the invention after single doseintravenous injections, and as described previously (Phillips G D L, LiG M, Dugger D L, et al. Targeting HER2-Positive Breast Cancer withTrastuzumab-DM1, an Antibody-Cytotoxic Drug Conjugate. (2008) CancerRes. 68:9280-90), incorporated by reference herein. Anti-Her2 ADC weretested with the Fo5 model, a transgenic mouse model in which the humanHER2 gene is over-expressed in mammary epithelium under transcriptionalregulation of the murine mammary tumor virus promoter (MMTV-HER2) asshown in FIGS. 3 and 5. The HER2 over-expression causes spontaneousdevelopment of a mammary tumor. The mammary tumor of one of thesefounder animals (founder #5 [Fo5]) has been propagated in subsequentgenerations of FVB mice by serial transplantation of tumor fragments(˜2×2 mm in size). All studies were conducted in accordance with theGuide for the Care and Use of Laboratory Animals. Each antibody-drugconjugate (single dose) was dosed in nine animals intravenously at thestart of the study, and 14 days post-transplant. Initial tumor size wasabout 200 mm³ volume. Measurements of tumor growth inhibition over timeby antibody-drug conjugates of the invention and controls are shown inFIGS. 3-5.

Another mammary fat pad transplant efficacy model may be employed asdescribed (Chen et al. (2007) Cancer Res 67:4924-4932), evaluating tumorvolume after a single intravenous dose and using tumors excised from amouse bearing an intraperitoneal tumor, then serially passaged into themammary fat pads of recipient mice.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. All patents, patent applications,and references cited throughout the specification are expresslyincorporated by reference.

We claim:
 1. A linker-drug intermediate of Formula I Ant-L-(Z)_(m)—X  I wherein Ant is selected from the structure:

where the wavy line indicates the attachment to L; L is a linker selected from —CH₂O—, —CH₂N(R)—, —N(R)—, —N(R)(C₁-C₁₂ alkylene)-, —N(R)(C₂-C₈ alkenylene)-, —N(R)(C₂-C₈ alkynylene)-, —N(R)(CH₂CH₂O)_(n)—, and the structure:

where the wavy lines indicate the attachments to Ant and Z; and Z is an optional spacer selected from —CH₂C(O)—, —CH₂C(O)NR(C₁-C₁₂ alkylene)-, and the structure:

where the wavy lines indicate the attachments to L and X; R is H, C₁-C₁₂ alkyl, or C₆-C₂₀ aryl; Z¹ is selected from —(C₁-C₁₂ alkylene)-, —(C₂-C₈ alkenylene)-, —(C₂-C₈ alkynylene)-, —O(C₁-C₁₂ alkylene)-, —O(C₂-C₈ alkenylene)-, —O(C₂-C₈ alkynylene)-, and (CH₂CH₂O)_(n)—; m is 0 or 1; n is 1 to 6; X is pyridyl disulfide where pyridyl is optionally substituted with one or more groups selected from NO₂, Cl, F, CN and Br; and alkylene, alkenylene, alkynylene, alkyl, and aryl are optionally substituted with one or more groups selected from F, Cl, Br, N(CH₃)₂, and OCH₃.
 2. The linker-drug intermediate of claim 1 where L is —CH₂O—.
 3. The linker-drug intermediate of claim 1 where L is —N(R)— or —N(R)(C₁-C₁₂ alkylene)-.
 4. The linker-drug intermediate of claim 1 wherein Z¹ is —(C₁-C₁₂ alkylene)- or —O(C₁-C₁₂ alkylene)-.
 5. The linker-drug intermediate of claim 1 where L is —CH₂O—; m is 1; and Z is


6. The linker-drug intermediate of claim 5 where Z¹ is —O(C₁-C₁₂ alkylene)-.
 7. The linker-drug intermediate of claim 5 where R is H or C₁-C₁₂ alkyl.
 8. The linker-drug intermediate of claim 1 wherein X is:

where the wavy line indicates the attachments to L or Z; R¹ is NO₂, Cl, F, CN or Br, and q is 0, 1, or
 2. 9. The linker-drug intermediate of claim 1 selected from Formulas Ia-c:

where R¹ is NO₂, Cl, F, or Br; and q is 0, 1, or
 2. 10. The linker-drug intermediate of claim 9 wherein R¹ is NO₂ and n is
 1. 11. The linker-drug intermediate of claim 1 having Formula Id:


12. The linker-drug intermediate of claim 1 selected from:


13. The linker-drug intermediate of claim 1 wherein X is:

where the wavy line indicates the attachments to L or Z.
 14. The linker-drug intermediate of claim 8 wherein X is:

where the wavy line indicates the attachments to L or Z.
 15. The linker-drug intermediate of claim 3 wherein L is selected from the group consisting of NHCH₂CH₂, NHCH₂C(CH₃)₂, and NHCH₂CH(CH₃); and m is
 0. 16. The linker-drug intermediate of claim 15 wherein L is selected from racemic NHCH₂CH(CH₃), the R stereoisomer of NHCH₂CH(CH₃), the S stereoisomer of NHCH₂CH(CH₃), and a mixture of the R and S stereoisomers of NHCH₂CH(CH₃).
 17. The linker-drug intermediate of claim 5 where Z is


18. The linker-drug intermediate of claim 17 where Z¹ is selected from the group consisting of —OCH₂CH₂—, —OCH₂C(CH₃)₂—, and —OCH₂CH(CH₃)—. 